Method for obtaining purified RNA from viable oocysts

ABSTRACT

The present invention describes novel oligonucleotides targeted to nucleic acid sequences derived from  Crytosporidium  organisms, and  Crytosporidium parvum  organisms in particular, which are useful for determining the presence of  Cryptosporidium  organisms in a test sample. The oligonucleotides of the present invention include hybridization assay probes, helper probes and amplification primers. The present invention further describes a novel method for obtaining purified ribonucleic acid from viable oocysts.

This application is a continuation of U.S. application Ser. No.09/954,586, filed Sep. 11, 2001, now U.S. Pat. No. 7,081,527, whichclaims the benefit of U.S. Provisional Application No. 60/232,028, filedSep. 12, 2000, the contents of which are hereby incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to hybridization assay probes, helperprobes, amplification primers, nucleic acid compositions, probe mixes,methods and kits useful for determining the presence of Cryptosporidiumorganisms in general, and Cryptosporidium parvum organisms inparticular, in a test sample of water, feces, food or other samplemedia.

The present invention further relates to a method for obtaining purifiedribonucleic acid from a viable oocyst (e.g., a Cryptosporidiumorganism). The purified ribonucleic acid may then be made available foramplification and/or detection using one or more amplificationoligonucleotides and/or a hybridization assay probe.

INCORPORATION BY REFERENCE

All references referred to herein are hereby incorporated by referencein their entirety. The incorporation of these references, standingalone, should not be construed as an assertion or admission by theinventors that any portion of the contents of all of these references,or any particular reference, is considered to be essential material forsatisfying any national or regional statutory disclosure requirement forpatent applications. Notwithstanding, the inventors reserve the right torely upon any of such references, where appropriate, for providingmaterial deemed essential to the claimed invention by an examiningauthority or court. No reference referred to herein is admitted to beprior art to the claimed invention.

BACKGROUND OF THE INVENTION

Cryptosporidium is an apicomplexan parasite capable of infecting avariety of animals, including humans. One particular species,Cryptosporidium parvum, is a coccidian parasite that poses particularthreat to humans by causing gasteroenteritis in immuno-competent adultsand infants, and occasionally life-threatening diarrhea inimmuno-deficient individuals, such as infants, the elderly and AIDSpatients. The mortality rate in AIDS patients infected withCryptosporidium parvum is as high as fifty percent. Transmission ofCryptosporidium parvum is direct, by the fecal-oral route or bycontamination of water supplies, swimming pools, and untreated surfacewater. Able to infect with as few as 30 microsopic oocysts,Cryptosporidium parvum is considered a leading cause of persistentdiarrhea in developing countries and is a major threat to the U.S. watersupply.

Cryptosporidium parvum exists in nature as a thick walled oocyst that isextremely resistant to environmental factors, exhibiting the ability tosurvive for months in the environment if maintained in a cool and moistsetting. The oocysts are highly resistant to conventional chlorinationof drinking water and their size presents a problem for water filtrationsystems typically employed by municipal water authorities. In fact,Cryptosporidium parvum oocysts are so robust that they are able tomaintain their integrity and viability after a 24 hour exposure to fullstrength bleach. Their resistance to destruction by environmental andchemical means is particularly alarming since nearly ninety percent ofall raw water supplies are infected with Cryptosporidium parvum oocysts.

In the United States, the method currently approved by the EnvironmentalProtection Agency for detecting Cryptosporidium parvum is an antibodystaining method referred to as “Method 1622”. This method has severaldrawbacks, including the subjectivity of the assay, high antibodybackground resulting in an unacceptable number of false positives, andits labor intensive aspects, requiring hours of microscopic analysis.Other methods currently available for detecting Cryptosporidium parvum,including detection of genomic DNA by the polymerase chain reaction(PCR) and immunological assays such as the enzyme-linked immunosorbentassay (ELISA), have significant sensitivity and specificity problems.Thus, a need exists for a sensitive and specific assay which can be usedto determine the presence of Cryptosporidium organisms, andCryptosporidium parvum in particular, in a test sample, as well as amethod for releasing the contents of the thick walled oocysts.

SUMMARY OF THE INVENTION

The present invention features oligonucleotides which are useful fordetermining whether organisms belonging to the genus Cryptosporidium orthe species Cryptosporidium parvum are present in a test sample such aswater, feces, food or other sample media. The featured oligonucleotidesmay be contained in amplification primers, hybridization assay probesand/or helper probes which are useful for amplifying and/or determiningwhether organisms belonging to the genus Cryptosporidium or the speciesCryptosporidium parvum are present in a test sample.

For instance, the hybridization assay probes can preferentiallyhybridize to a target region present in nucleic acid derived fromCryptosporidium organisms to form a detectable probe:target hybridindicating the presence of organisms belonging to the Cryptosporidiumgenus, which may include such species as Cryptosporidium agni,Cryptosporidium baileyi, Cryptosporidium bovis, Cryptosporidium crotali,Cryptosporidium meleagridis, Cryptosporidium muris, Cryptosporidiumnasorum, Cryptosporidium parvum, Cryptosporidium serpentis and/orCryptosporidium wrairi, as well as other organisms belonging to theCryptosporidium genus. In one embodiment, the invention provideshybridization assay probes for determining whether Cryptosporidiumorganisms are present in a test sample, which probes preferably containan at least 10 contiguous base region which is at least 80%complementary (preferably at least 90% complementary, and morepreferably 100% complementary) to an at least 10 contiguous base regionpresent in a target nucleic acid sequence present in a target nucleicacid derived from Cryptosporidium organisms, which target nucleic acidsequence is selected from the group consisting of (reading 5′ to 3′):

SEQ ID NO:1: ctatcagctttagacggtaggg, SEQ ID NO:2:cuaucagcuuuagacgguaggg, SEQ ID NO:3: ccctaccgtctaaagctgatag, and SEQ IDNO:4: cccuaccgucuaaagcugauag.These probes will preferentially hybridize to the target nucleic acidand not to nucleic acid derived from non-Cryptosporidium organisms understringent hybridization assay conditions.

In another embodiment, the invention provides hybridization assay probesfor determining whether organisms of the species Cryptosporidium parvumare present in a test sample, which probes preferably contain an atleast 10 contiguous base region which is at least 80% complementary(preferably at least 90% complementary, and more preferably 100%complementary) to an at least 10 contiguous base region present in atarget nucleic acid sequence present in a target nucleic acid derivedfrom Cryptosporidium parvum organisms, which target nucleic acidsequence is selected from the group consisting of (reading 5′ to 3′):

SEQ ID NO:5: gcgaaaaaactcgactttatggaaggg, SEQ ID NO:6:aactcgactttatggaaggg, SEQ ID NO:7: aaaactcgactttatggaagggttg, SEQ IDNO:8: gttaaagacaaactaatgcgaaagc, SEQ ID NO:9:gcgaaaaaacucgacuuuauggaaggg, SEQ ID NO:10: aacucgacuuuauggaaggg, SEQ IDNO:11: aaaacucgacuuuauggaaggguug, SEQ ID NO:12:guuaaagacaaacuaaugcgaaagc, SEQ ID NO:13: cccttccataaagtcgagttttttcgc,SEQ ID NO:14: cccttccataaagtcgagtt, SEQ ID NO:15:caacccttccataaagtcgagtttt, SEQ ID NO:16: gctttcgcattagtttgtctttaac, SEQID NO:17: cccuuccauaaagucgaguuuuuucgc, SEQ ID NO:18:cccuuccauaaagucgaguu, SEQ ID NO:19: caacccuuccauaaagucgaguuuu, and SEQID NO:20: gcuuucgcauuaguuugucuuuaac.These probes will preferentially hybridize to the target nucleic acidand not to nucleic acid derived from non-Cryptosporidium parvumorganisms (especially Cryptosporidium muris, Cryptosporidium baileyi andCryptosporidium wrairi) present in the test sample under stringenthybridization assay conditions.

Preferably, the Cryptosporidium and Cryptosporidium parvum probes of thepresent invention comprise an oligonucleotide up to 100 bases in length(preferably from 12 to 50 bases, and more preferably from 18 to 35 basesin length) and substantially complementary to the target nucleic acidsequence (preferably perfectly complementary to the first target nucleicacid sequence). The oligonucleotide may consist of deoxyribonucleic acid(DNA), ribonucleic acid (RNA), a combination DNA and RNA, or it may be anucleic acid analog (e.g., a peptide nucleic acid) or contain one ormore modified nucleosides (e.g., a ribonucleoside having a 2′-O-methylsubstitution to the ribofuranosyl moiety).

The probes preferably include a detectable label. The label may be anysuitable labeling substance, including but not limited to aradioisotope, an enzyme, an enzyme cofactor, an enzyme substrate, a dye,a hapten, a chemiluminescent molecule, a fluorescent molecule, aphosphorescent molecule, an electrochemiluminescent molecule, achromophore, a base sequence region that is unable to stably hybridizeto the target nucleic acid under the stated conditions, and mixtures ofthese. In one particularly preferred embodiment, the label is anacridinium ester.

In another embodiment, the invention contemplates probe mixes that areuseful for determining whether Cryptosporidium organisms, orCryptosporidium parvum organisms in particular, are present in a testsample. For instance, to determine the presence of organisms from thegenus Cryptosporidium, the probe mix may comprise one of theabove-described Cryptosporidium probes and a helper probe. Preferably,the helper probe is an oligonucleotide up to 100 bases in length, morepreferably from 12 to 50 bases in length, and even more preferably from18 to 35 bases in length. Preferably, the helper probe contains an atleast 10 contiguous base region which is at least 80% complementary(preferably at least 90% complementary, and more preferably 100%complementary) to an at least 10 contiguous base region present in atarget nucleic acid sequence present in a target nucleic acid derivedfrom Cryptosporidium organisms, which target sequence is selected fromthe group consisting of (reading 5′ to 3′):

SEQ ID NO:21: gacatatcattcaagtttctgac, SEQ ID NO:22:ttggcctaccgtggcaatgacggg, SEQ ID NO:23: gacauaucauucaaguuucugac, SEQ IDNO:24: uuggccuaccguggcaaugacggg, SEQ ID NO:25: gtcagaaacttgaatgatatgtc,SEQ ID NO:26: cccgtcattgccacggtaggccaa, SEQ ID NO:27:gucagaaacuugaaugauauguc, SEQ ID NO:28: cccgucauugccacgguaggccaa,and mixtures thereof. The helper probes may be, but need not be,perfectly complementary to the target sequence.

To determine the presence of Cryptosporidium parvum organisms in a testsample, the probe mix may comprise one of the above-describedCryptosporidium parvum probes and a helper probe. Preferably, the helperprobe is an oligonucleotide up to 100 bases in length, more preferablyfrom 12 to 50 bases, and even more preferably from 18 to 35 bases inlength. Preferably, the helper probe contains an at least 10 contiguousbase region which is at least 80% complementary (preferably at least 90%complementary, and more preferably 100% complementary) to an at least 10contiguous base region present in a target nucleic acid sequence presentin a target nucleic acid derived from Cryptosporidium parvum organisms,which target sequence is selected from the group consisting of (reading5′ to 3′):

SEQ ID NO:29: ggataaccgtggtaattctagagctaatacat, SEQ ID NO:30:ccgtggtaattctagagctaatacat, SEQ ID NO:31: ttgtatttattagataaagaacc, SEQID NO:32: ttgtatttattagataaagaaccaatata, SEQ ID NO:33:ggauaaccgugguaauucuagagcuaauacau, SEQ ID NO:34:ccgugguaauucuagagcuaauacau, SEQ ID NO:35: uuguauuuauuagauaaagaacc, SEQID NO:36: uuguauuuauuagauaaagaaccaauaua, SEQ ID NO:37:atgtattagctctagaattaccacggttatcc, SEQ ID NO:38:atgtattagctctagaattaccacgg, SEQ ID NO:39: ggttctttatctaataaatacaa, SEQID NO:40: tatattggttctttatctaataaatacaa, SEQ ID NO:41:auguauuagcucuagaauuaccacgguuaucc, SEQ ID NO:42:auguauuagcucuagaauuaccacgg, SEQ ID NO:43: gguucuuuaucuaauaaauacaa, SEQID NO:44: uauauugguucuuuaucuaauaaauacaa,and mixtures thereof. The helper probes may be, but need not be,perfectly complementary to the target sequence.

In a preferred embodiment for the Cryptosporidium parvum probe mix, thehybridization assay probe comprises an oligonucleotide having an atleast 10 contiguous base region which is at least 80% homologous to anat least 10 contiguous base region present in a sequence selected fromthe group consisting of: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:17 and SEQ ID NO:18; andthe helper probe comprises an oligonucleotide having an at least 10contiguous base region which is at least 80% homologous to an at least10 contiguous base region present in a sequence selected from the groupconsisting of: SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,SEQ ID NO:43 and SEQ ID NO:44.

In another preferred embodiment for the Cryptosporidium parvum probemix, the hybridization assay probe comprises an oligonucleotide havingan at least 10 contiguous base region which is at least 80% homologousto an at least 10 contiguous base region present in a sequence selectedfrom the group consisting of: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:17 and SEQ ID NO:18; andthe probe mix comprises first and second helper probes, where the firsthelper probe comprises an oligonucleotide having an at least 10contiguous base region which is at least 80% homologous to an at least10 contiguous base region present in a sequence selected from the groupconsisting of: SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:41 and SEQ ID NO:42, and where thesecond helper probe comprises an oligonucleotide having an at least 10contiguous base region which is at least 80% homologous to an at least10 contiguous base region present in a sequence selected from the groupconsisting of: SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:43 and SEQ ID NO:44.

In another preferred embodiment for the Cryptosporidium parvum probemix, the hybridization assay probe comprises an oligonucleotide havingan at least 10 contiguous base region which is at least 80% homologousto an at least 10 contiguous base region present in a sequence selectedfrom the group consisting of: SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15and SEQ ID NO:19; and the helper probe comprises an oligonucleotidehaving an at least 10 contiguous base region which is at least 80%homologous to an at least 10 contiguous base region present in asequence selected from the group consisting of: SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:41 and SEQ ID NO:42.

The invention also contemplates compositions comprising stable nucleicacid duplexes formed between the above-described hybridization assayprobes and/or helper probes and the target nucleic acids for the probesunder stringent hybridization assay conditions.

The invention also features amplification primers useful for detectingthe presence of Cryptosporidium organisms in an amplification assay. Inone preferred embodiment, the invention provides at least oneamplification primer for amplifying nucleic acid derived fromCryptosporidium organisms present in a test sample, which amplificationprimer preferably contains an at least 10 contiguous base region whichis at least 80% complementary (preferably at least 90% complementary,and more preferably 100% complementary) to an at least 10 contiguousbase region present in a target nucleic acid sequence present in atarget nucleic acid derived from Cryptosporidium organisms, which targetnucleic acid sequence is selected from the group consisting of (reading5′ to 3′):

SEQ ID NO:45: gccatgcatgtctaagtataaac, SEQ ID NO:46:ggataaccgtggtaattctagag, SEQ ID NO:47: ggtgactcataataactttacgg, SEQ IDNO:48: ctaccacatctaaggaaggcag, SEQ ID NO:49: gtatttaacagtcagaggtg, SEQID NO:50: gccaaggatgttttcattaatc, SEQ ID NO:51: gccaugcaugucuaaguauaaac,SEQ ID NO:52: ggauaaccgugguaauucuagag, SEQ ID NO:53:ggugacucauaauaacuuuacgg, SEQ ID NO:54: cuaccacaucuaaggaaggcag, SEQ IDNO:55: guauuuaacagucagaggug, SEQ ID NO:56: gccaaggauguuuucauuaauc, SEQID NO:57: gtttatacttagacatgcatggc, SEQ ID NO:58:ctctagaattaccacggttatcc, SEQ ID NO:59: ccgtaaagttattatgagtcacc, SEQ IDNO:60: ctgccttccttagatgtggtag, SEQ ID NO:61: cacctctgactgttaaatac, SEQID NO:62: gattaatgaaaacatccttggc, SEQ ID NO:63: guuuauacuuagacaugcauggc,SEQ ID NO:64: cucuagaauuaccacgguuaucc, SEQ ID NO:65:ccguaaaguuauuaugagucacc, SEQ ID NO:66: cugccuuccuuagaugugguag, SEQ IDNO:67: caccucugacuguuaaauac, and SEQ ID NO:68: gauuaaugaaaacauccuuggc.The amplification primers optionally include a 5′ sequence which isrecognized by a RNA polymerase or which enhances initiation orelongation by RNA polymerase. When included, a T7 promoter, such as SEQID NO:69 aatttaatacgactcactatagggaga, is preferred.

The invention further contemplates amplification primers which, whencontacted with a nucleic acid polymerase under amplification conditions,will bind to or cause extension through a nucleic acid region having abase sequence selected from the group consisting of: SEQ ID NO:45, SEQID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67 and SEQ ID NO:68. The amplification primers of thisembodiment also optionally include a 5′ sequence which is recognized bya RNA polymerase or which enhances initiation or elongation by RNApolymerase. The T7 promoter of SEQ ID NO:69 is preferred.

Amplification primers of the present invention are preferably employedin sets of at least two amplification primers. Preferred sets include afirst amplification primer which contains an at least 10 contiguous baseregion which is at least 80% complementary to an at least 10 contiguousbase region present in a sequence selected from the group consisting of:SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52,SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:58; SEQ ID NO:61, SEQ ID NO:63,SEQ ID NO:64 and SEQ ID NO:67. The second amplification primer of thesepreferred sets contains an at least 10 contiguous base region which isat least 80% complementary to an at least 10 contiguous base regionpresent in a sequence selected from the group consisting of: SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:59, SEQ ID NO:60; SEQ ID NO:62, SEQ ID NO:65, SEQ IDNO:66 and SEQ ID NO:68.

The invention additionally contemplates compositions comprising stablenucleic acid duplexes formed between the above-described amplificationprimers and the target nucleic acids for the primers under amplificationconditions.

The invention further features methods for determining whetherCryptosporidium or Cryptosporidium parvum organisms are present in atest sample. In one embodiment, the invention provides a method fordetermining whether Cryptosporidium organisms are present in a testsample, where the method comprises the steps of: (a) contacting the testsample with one of the above-described hybridization assay probes fordetecting Cryptosporidium organisms under conditions permitting theprobe to preferentially hybridize to a target nucleic acid derived froma Cryptosporidium organism, thereby forming a probe:target hybrid stablefor detection; and (b) determining whether the hybrid is present in thetest sample as an indication of the presence or absence ofCryptosporidium organisms in the test sample. This method may furtherinclude the step of quantifying the amount of hybrid present in the testsample as a means for estimating the amount of Cryptosporidium organismspresent in the test sample.

In another embodiment, the invention provides a method for determiningwhether Cryptosporidium parvum organisms are present in a test sample,where the method comprises the steps of: (a) contacting the test samplewith one of the above-described hybridization assay probes for detectingCryptosporidium parvum organisms under conditions permitting the probeto preferentially hybridize to a target nucleic acid derived from aCryptosporidium parvum organism, thereby forming a probe:target hybridstable for detection; and (b) determining whether the hybrid is presentin the test sample as an indication of the presence or absence ofCryptosporidium parvum organisms in the test sample. This method mayfurther include the step of quantifying the amount of hybrid present inthe test sample as a means for estimating the amount of Cryptosporidiumparvum organisms present in the test sample.

The methods for determining whether Cryptosporidium or Cryptosporidiumparvum organisms are present in a test sample, or the amount of theseorganisms present in a test sample, may further include the step ofcontacting the test sample with at least one of the above-describedhelper probes, appropriate for Cryptosporidium or Cryptosporidiumparvum, as desired. (Preferred hybridization assay probe and helperprobe combinations are set forth above as probe mixes.) In addition tothe helper probes, or in the alternative, the methods may furtherinclude the step of contacting the test sample with at least one of theabove-described amplification primers, appropriate for amplifying atarget nucleic acid sequence present in nucleic acid derived fromCryptosporidium or Cryptosporidium parvum organisms, as desired.

The invention also contemplates methods for amplifying a target nucleicacid sequence present in nucleic acid derived from a Cryptosporidiumorganism present in a test sample, where the method comprises the stepsof: (a) contacting the test sample with at least one of theabove-described amplification primers under amplification conditions;and (b) amplifying the target nucleic acid sequence. The target nucleicacid sequence amplified may be present in nucleic acid derived fromCryptosporidium organisms or, in particular, from Cryptosporidium parvumorganisms. Preferred amplification methods will include a set of atleast two of the above-described amplification primers.

In one embodiment, the method for amplifying a target nucleic acidsequence present in nucleic acid derived from a Cryptosporidium organismwill further include the steps of: (a) contacting the test sample with ahybridization assay probe which preferentially hybridizes to the targetnucleic acid sequence, or a complement thereof, under stringenthybridization conditions, thereby forming a probe:target hybrid stablefor detection; and (b) determining whether the hybrid is present in thetest sample as an indication of the presence or absence ofCryptosporidium or Cryptosporidium parvum organisms in the test sample.The above-described hybridization assay probes are especially preferredfor this method.

The invention also contemplates kits for determining whetherCryptosporidium organisms are present in a test sample. These kitscomprise at least one of the above-described hybridization assay probesspecific for Cryptosporidium-derived nucleic acid and optionally includewritten instructions for determining the presence or amount ofCryptosporidium organisms in a test sample. In another embodiment, thekits further comprise at least one of the above-described helper probesappropriate for nucleic acid derived from Cryptosporidium. In a furtherembodiment, the kits also comprise at least one of the above-describedamplification primers appropriate for amplifying a target nucleic acidsequence present in nucleic acid derived from Cryptosporidium organisms.In yet another embodiment, the kits further comprise at least one of theabove-described helper probes and at least one of the above-describedamplification primers.

Similarly, the invention contemplates kits for determining whetherCryptosporidium parvum organisms are present in a test sample. Thesekits comprise at least one of the above-described hybridization assayprobes specific for Cryptosporidium parvum-derived nucleic acid andoptionally include written instructions for determining the presence oramount of Cryptosporidium parvum organisms in a test sample. In anotherembodiment, the kits further comprise at least one of theabove-described helper probes appropriate for nucleic acid derived fromCryptosporidium parvum organisms. In a further embodiment, the kits alsocomprise at least one of the above-described amplification primersappropriate for amplifying a target nucleic acid sequence present innucleic acid derived from Cryptosporidium parvum organisms. In anotherembodiment, the kits comprise at least one of the above-described helperprobes and at least one of the above-described amplification primers.

The invention also contemplates kits for amplifying a target nucleicacid sequence present in nucleic acid derived from Cryptosporidium orCryptosporidium parvum organisms, comprising at least one of theabove-described amplification primers and optionally include writteninstructions for amplifying nucleic acid derived from Cryptosporidium orCryptosporidium parvum organisms, as appropriate.

Those skilled in the art will appreciate that the hybridization assayprobes of the present invention may be used as amplification primers orcapture probes, the amplification primers of the present invention maybe used as hybridization assay probes or capture probes, and the helperprobes of the present invention may be used as amplification primers orcapture probes, depending upon the degree of specificity required.

The present invention further features a method for obtaining purifiedRNA from a viable oocyst, such as a Cryptosporidium organism.Ribonucleic acid isolated from an oocyst may then be made available foramplification and/or detection using at least one of the above-describedamplification primers and/or hybridization assay probes.

In a preferred embodiment, the method for obtaining purified RNA from aviable oocyst includes the steps of: (i) centrifuging a fluid samplesuspected of containing oocysts at a speed and for a period of timesufficient to concentrate the oocysts within a vessel containing thefluid sample; (ii) removing a supernatant from the vessel which formedduring the centrifuging step; (iii) resuspending the concentratedoocysts, if present, in a buffered solution; (iv) oscillating, orotherwise agitating (e.g., vortexing), the buffered solution in thepresence of a plurality of particles at a rate and for a period of timesufficient to lyse the oocysts and release RNA therefrom; (v)immobilizing the released RNA on an RNA-binding filter (e.g., asilica-based matrix); (vi) purifying the released RNA by washing thefilter one or more times with a buffered solution to remove oocystcomponents (e.g., proteins, DNA and other contaminants) other than thereleased RNA; and (vii) removing the purified RNA from the filter. Thebuffered solution used in step (iii) of this method preferably includesa chaotropic agent, such as guanidinium thiocyanate, which inactivatesendogenous ribonucleases released from the oocysts. The particles usedto lyse the oocysts may be, for example, glass, zirconia-glass,zirconia, stainless steel, chrome-steel or tungsten carbide, althoughzirconia-glass particles having a density of about 3.7 g/cc arepreferred. The particles preferably have a generally spherical shape andpreferably have an average diameter in the range of about 0.1 to about2.5 mm, more preferably in the range of about 0.5 to about 1.0 mm, andmost preferably have an average diameter of about 1.0 mm.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an oocyst titration graph plotting “Average Net RLU” versus“Oocysts” determined by hemocytometer counting. This figure indicatesthe oocyst load necessary to directly detect the presence ofCryptosporidium parvum in a test sample.

FIG. 2 is an amplification graph plotting “Average Net RLU” versus“Target.” This figure provides an indication of the initial rRNA copynumber needed to detect the presence of Cryptosporidium parvum organismsin a test sample following a transcription-mediated amplificationprocedure.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention describes oligonucleotides targeted to nucleicacids derived from Cryptosporidium organisms which are particularlyuseful for determining the presence or absence of Cryptosporidiumorganisms generally, and Cryptosporidium parvum organisms in particular,in a test sample. The oligonucleotides can aid in detectingCryptosporidium organisms in different ways, such as by functioning ashybridization assay probes, helper probes and/or amplification primers.Hybridization assay probes of the present invention can preferentiallyhybridize to a target nucleic acid sequence present in a target nucleicacid derived from Cryptosporidium organisms under stringenthybridization assay conditions to form detectable duplexes whichindicate the presence of Cryptosporidium organisms, or more specificallyCryptosporidium parvum organisms, in a test sample. Some of the probesare believed to be capable of distinguishing between Cryptosporidium andits known closest phylogenetic neighbors. Other of the probes arebelieved to be capable of distinguishing between Cryptosporidium parvumand its known closest phylogenetic neighbors. Helper probes of thepresent invention can hybridize to a target nucleic acid sequencepresent in nucleic acid derived from Cryptosporidium organisms understringent hybridization assay conditions and can be used to enhance theformation of hybridization assay probe:target nucleic acid duplexes.Amplification primers of the present invention can hybridize to a targetnucleic acid sequence present in nucleic acid derived fromCryptosporidium organisms under amplification conditions and can be usedas primers in amplification reactions to generate Cryptosporidium, ormore specifically Cryptosporidium parvum, derived nucleic acid. Theprobes and amplification primers may be used in assays for the detectionand/or quantitation of Cryptosporidium or Cryptosporidium parvumorganisms in a test sample.

The present invention further describes a method for obtaining purifiedRNA from a viable oocyst, such as a Cryptosporidium organism. Thepurified RNA may be made available for amplification with one or moreamplification primers and/or detection with a hybridization assay probe.

A. Definitions

The following terms have the indicated meanings in the specificationunless expressly indicated to have a different meaning.

By “target nucleic acid” or “target” is meant a nucleic acid containinga target nucleic acid sequence.

By “target nucleic acid sequence,” “target nucleotide sequence,” “targetsequence” or “target region” is meant a specific deoxyribonucleotide orribonucleotide sequence comprising all or part of the nucleotidesequence of a single-stranded nucleic acid molecule.

By “oligonucleotide” or “oligomer” is meant a polymer made up of two ormore nucleoside subunits or nucleobase subunits coupled together. Theoligonucleotide may be DNA and/or RNA and analogs thereof. The sugargroups of the nucleoside subunits may be ribose, deoxyribose and analogsthereof, including, for example, ribonucleosides having a 2′-O-methylsubstitution to the ribofuranosyl moiety. (Oligonucleotides includingnucleoside subunits having 2′ substitutions and which are useful ashybridization assay probes, helper probes and/or amplification primersare disclosed by Becker et al., “Method for Amplifying Target NucleicAcids Using Modified Primers,” U.S. Pat. No. 6,130,038.) The nucleosidesubunits may by joined by linkages such as phosphodiester linkages,modified linkages or by non-nucleotide moieties which do not preventhybridization of the oligonucleotide to its complementary target nucleicacid sequence. Modified linkages include those linkages in which astandard phosphodiester linkage is replaced with a different linkage,such as a phosphorothioate linkage or a methylphosphonate linkage. Thenucleobase subunits may be joined, for example, by replacing the naturaldeoxyribose phosphate backbone of DNA with a pseudo peptide backbone,such as a 2-aminoethylglycine backbone which couples the nucleobasesubunits by means of a carboxymethyl linker to the central secondaryamine. (DNA analogs having a pseudo peptide backbone are commonlyreferred to as “peptide nucleic acids” or “PNA” and are disclosed byNielsen et al., “Peptide Nucleic Acids,” U.S. Pat. No. 5,539,082.) Othernon-limiting examples of oligonucleotides or oligomers contemplated bythe present invention include nucleic acid analogs containing bicyclicand tricyclic nucleoside and nucleotide analogs referred to as “LockedNucleic Acids,” “Locked Nucleoside Analogues” or “LNA.” (Locked NucleicAcids are disclosed by Wang, “Conformationally Locked Nucleosides andOligonucleotide,” U.S. Pat. No. 6,083,482; Imanishi et al., “NovelBicyclonucleoside and Oligonucleotide Analogues,” InternationalPublication No. WO 98/39352; and Wengel et al., “OligonucleotideAnalogues,” International Publication No. WO 99/14226.) Any nucleic acidanalog is contemplated by the present invention provided the modifiedoligonucleotide can hybridize to a target nucleic acid under stringenthybridization assay conditions or amplification conditions. In the caseof hybridization assay probes, the modified oligonucleotides must alsobe capable of preferentially hybridizing to the target nucleic acidunder stringent hybridization assay conditions.

Oligonucleotides of a defined sequence may be produced by techniquesknown to those of ordinary skill in the art, such as by chemical orbiochemical synthesis, and by in vitro or in vivo expression fromrecombinant nucleic acid molecules, e.g., bacterial or retroviralvectors. As intended by this disclosure, an oligonucleotide does notconsist of wild-type chromosomal DNA or the in vivo transcriptionproducts thereof. One use of an oligonucleotide is as a hybridizationassay probe. Oligonucleotides may also be used as in vivo or in vitrotherapeutic amplification primers or as antisense agents to block orinhibit gene transcription or translation in diseased, infected, orpathogenic cells.

By “hybridization assay probe” or “probe” is meant an oligonucleotidehaving a base sequence sufficiently complementary to its target nucleicacid sequence to form a probe:target hybrid stable for detection understringent hybridization assay conditions. As would be understood bysomeone having ordinary skill in the art, a probe is an isolated nucleicacid molecule, or an analog thereof, in a form not found in naturewithout human intervention (e.g., recombined with foreign nucleic acid,isolated, or purified to some extent). The probes of this invention mayhave additional nucleosides or nucleobases outside of the targetedregion so long as such nucleosides or nucleobases do not preventhybridization under stringent hybridization conditions and, in the caseof hybridization assay probes, do not prevent preferential hybridizationto the target nucleic acid. A non-complementary sequence may also beincluded, such as a target capture sequence (generally a homopolymertract, such as a poly-A, poly-T or poly-U tail), promotor sequence, abinding site for RNA transcription, a restriction endonucleaserecognition site, or sequences which will confer a desired secondary ortertiary structure, such as a catalytic active site or a hairpinstructure, which can be used to facilitate detection and/oramplification. Probes of a defined sequence may be produced bytechniques known to those of ordinary skill in the art, such as bychemical synthesis, and by in vitro or in vivo expression fromrecombinant nucleic acid molecules.

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex. The temperature of the reaction mixture is morepreferably at least 5° C. below the melting temperature of the nucleicacid duplex, and even more preferably at least 10° C. below the meltingtemperature of the reaction mixture.

By “substantially homologous,” “substantially corresponding” or“substantially corresponds” is meant that the subject oligonucleotidehas a base sequence containing an at least 10 contiguous base regionthat is at least 80% homologous, preferably at least 90% homologous, andmost preferably 100% homologous to an at least 10 contiguous base regionpresent in a reference base sequence (excluding RNA and DNAequivalents). (Those skilled in the art will readily appreciatemodifications that could be made to the hybridization assay conditionsat various percentages of homology to permit hybridization of theoligonucleotide to the target sequence while preventing unacceptablelevels of non-specific hybridization.) The degree of similarity isdetermined by comparing the order of nucleobases making up the twosequences and does not take into consideration other structuraldifferences which may exist between the two sequences, provided thestructural differences do not prevent hydrogen bonding withcomplementary bases. The degree of homology between two sequences canalso be expressed in terms of the number of base mismatches present ineach set of at least 10 contiguous bases being compared, which may rangefrom 0 to 2 base differences.

By “substantially complementary” is meant that the subjectoligonucleotide has a base sequence containing an at least 10 contiguousbase region that is at least 80% complementary, preferably at least 90%complementary, and most preferably 100% complementary to an at least 10contiguous base region present in a target nucleic acid sequence(excluding RNA and DNA equivalents). (Those skilled in the art willreadily appreciate modifications that could be made to the hybridizationassay conditions at various percentages of complementarity to permithybridization of the oligonucleotide to the target sequence whilepreventing unacceptable levels of non-specific hybridization.) Thedegree of complementarity is determined by comparing the order ofnucleobases making up the two sequences and does not take intoconsideration other structural differences which may exist between thetwo sequences, provided the structural differences do not preventhydrogen bonding with complementary bases. The degree of complementaritybetween two sequences can also be expressed in terms of the number ofbase mismatches present in each set of at least 10 contiguous basesbeing compared, which may range from 0 to 2 base mismatches.

By “RNA and DNA equivalents” is meant RNA and DNA molecules having thesame complementary base pair hybridization properties. RNA and DNAequivalents have different sugar moieties (i.e., ribose versusdeoxyribose) and may differ by the presence of uracil in RNA and thyminein DNA. The differences between RNA and DNA equivalents do notcontribute to differences in homology because the equivalents have thesame degree of complementarity to a particular sequence.

By “hybridization” is meant the ability of two completely or partiallycomplementary nucleic acid strands to come together under specifiedhybridization assay conditions in a parallel or preferably antiparallelorientation to form a stable structure having a double-stranded region.The two constituent strands of this double-stranded structure, sometimescalled a hybrid, are held together by hydrogen bonds. Although thesehydrogen bonds most commonly form between nucleotides containing thebases adenine and thymine or uracil (A and T or U) or cytosine andguanine (C and G) on single nucleic acid strands, base pairing can alsoform between bases which are not members of these “canonical” pairs.Non-canonical base pairing is well-known in the art. (See, e.g., ROGERL. P. ADAMS ET AL., THE BIOCHEMISTRY OF THE NUCLEIC ACIDS (11^(th) ed.1992).)

By “preferentially hybridize” is meant that under stringenthybridization assay conditions, hybridization assay probes can hybridizeto their target nucleic acids to form stable probe:target hybridsindicating the presence of at least one organism of interest, and thereis not formed a sufficient number of stable probe:non-target hybrids toindicate the presence of non-targeted organisms, especiallyphylogenetically closely related organisms. Thus, the probe hybridizesto target nucleic acid to a sufficiently greater extent than tonon-target nucleic acid to enable one having ordinary skill in the artto accurately detect the presence (or absence) of nucleic acid derivedfrom Cryptosporidium or Cryptosporidium parvum, as appropriate, anddistinguish its presence from that of a phylogenetically closely relatedorganism in a test sample. In general, reducing the degree ofcomplementarity between an oligonucleotide sequence and its targetsequence will decrease the degree or rate of hybridization of theoligonucleotide to its target region. However, the inclusion of one ormore non-complementary nucleosides or nucleobases may facilitate theability of an oligonucleotide to discriminate against non-targetorganisms.

Preferential hybridization can be measured using techniques known in theart and described herein, such as in the examples provided below.Preferably, there is at least a 10-fold difference between target andnon-target hybridization signals in a test sample, more preferably atleast a 100-fold difference, and most preferably at least a 1,000-folddifference. Preferably, non-target hybridization signals in a testsample are no more than the background signal level.

By “s ngenthybridization assay conditions,” “hybridization assayconditions,” “stringent hybridization conditions,” or “stringentconditions” is meant conditions permitting a hybridization assay probeto preferentially hybridize to a target nucleic acid (preferably rRNA orrDNA derived from Cryptosporidium or Cryptosporidium parvum organisms)and not to nucleic acid derived from a closely related non-targetmicroorganism. Stringent hybridization assay conditions may varydepending upon factors including the GC content and length of the probe,the degree of similarity between the probe sequence and sequences ofnon-target sequences which may be present in the test sample, and thetarget sequence. Hybridization conditions include the temperature andthe composition of the hybridization reagents or solutions. While theExamples section infra provides preferred hybridization assay conditionsfor detecting target nucleic acids derived from Cryptosporidium orCryptosporidium parvum organisms using the probes of the presentinvention, other acceptable stringent conditions could be easilyascertained by someone having ordinary skill in the art.

By “consists essentially of” or “consisting essentially of,” when usedwith reference to an oligonucleotide herein, is meant that theoligonucleotide has a base sequence substantially homologous to aspecified base sequence and may have up to four additional bases and/ortwo bases deleted therefrom. Thus, these phrases contain both a sequencelength limitation and a sequence variation limitation. Any additions ordeletions are non-material variations of the specified base sequencewhich do not prevent the oligonucleotide from having its claimedproperty, such as being able to preferentially hybridize under stringenthybridization assay conditions to its target nucleic acid overnon-target nucleic acids. The oligonucleotide may contain a basesequence substantially similar to a specified nucleic acid sequencewithout any additions or deletions. However, a probe or primercontaining an oligonucleotide consisting essentially of (or whichconsists essentially of) a specified base sequence may include othernucleic acid molecules which do not participate in hybridization of theprobe to the target nucleic acid and which do not affect suchhybridization.

By “nucleic acid duplex,” “duplex,” “nucleic acid hybrid” or “hybrid” ismeant a stable nucleic acid structure comprising a double-stranded,hydrogen-bonded region. Such hybrids include RNA:RNA, RNA:DNA andDNA:DNA duplex molecules and analogs thereof. The structure issufficiently stable to be detectable by any known means, including meanswhich do not require a probe associated label. For instance, thedetection method may include a probe coated substrate which is opticallyactive and sensitive to changes in mass at its surface. Mass changesresult in different reflective and transmissive properties of theoptically active substrate in response to light and serve to indicatethe presence or amount of immobilized target nucleic acid. (Thisexemplary form of optical detection is disclosed by Nygren et aL.,“Devices and Methods for Optical Detection of Nucleic AcidHybridization,” U.S. Pat. No. 6,060,237.)

By “amplification primer” or “primer” is meant an oligonucleotidecapable of hybridizing to a target nucleic acid and acting as a primerand/or a promoter template (e.g., for synthesis of a complementarystrand, thereby forming a functional promoter sequence) for theinitiation of nucleic acid synthesis. If the amplification primer isdesigned to initiate RNA synthesis, the primer may contain a basesequence which is non-complementary to the target sequence but which isrecognized by an RNA polymerase such as a T7, T3 or SP6 RNA polymerase.An amplification primer may contain a 3′ terminus which is modified toprevent or lessen the rate or amount of primer extension. (McDonough etal., “Methods of Amplifying Nucleic Acids Using Promoter-ContainingPrimer Sequence,” U.S. Pat. No. 5,766,849, disclose primers andpromoter-primers having modified or blocked 3′-ends.) While theamplification primers of the present invention may be chemicallysynthesized or derived from a vector, they are not naturally-occurringnucleic acid molecules.

By “nucleic acid amplification” or “target amplification” is meantincreasing the number of nucleic acid molecules having at least onetarget nucleic acid sequence. Target amplification according to thepresent invention may be either linear or exponential, althoughexponential amplification is preferred.

By “amplification conditions” is meant conditions permitting nucleicacid amplification. While the Examples section infra provides preferredamplification conditions for amplifying target nucleic acid sequencesderived from Cryptosporidium or Cryptosporidium parvum organisms usingprimers of the present invention in a transcription-based method ofamplification, other acceptable amplification conditions could be easilyascertained by someone having ordinary skill in the art depending on theparticular method of amplification employed.

By “antisense,” “opposite sense” or “negative sense” is meant a nucleicacid molecule perfectly complementary to a reference, or sense, nucleicacid molecule.

By “sense,” “same-sense” or “positive sense” is meant a nucleic acidmolecule perfectly homologous to a reference nucleic acid molecule.

By “amplicon” is meant a nucleic acid molecule generated in a nucleicacid amplification reaction and which is derived from a target nucleicacid. An amplicon contains a target nucleic acid sequence which may beof the same or opposite sense as the target nucleic acid.

By “derived” is meant that the referred to nucleic acid is obtaineddirectly from an organism or is the product of a nucleic acidamplification. Thus, a nucleic acid which is “derived” from an organismmay be, for example, an antisense RNA molecule which does not naturallyexist in the organism.

By “capture probe” is meant one or more oligonucleotides linked togetherwhich are capable of hybridizing to a target nucleic acid—in a regionother than that targeted by a hybridization assay probe—and to animmobilized probe, thereby providing means for immobilizing andisolating the target nucleic acid in a test sample. The capture probeincludes both a target binding region, which hybridizes to the targetnucleic acid, and an immobilized probe binding region, which hybridizesto the immobilized probe. While the capture probe hybridizes to both thetarget nucleic acid and the immobilized probe under stringentconditions, the target binding and the immobilized probe binding regionsof the capture probe may be designed to bind to their target sequencesunder different hybridization conditions. In this way, the capture probemay be designed so that it first hybridizes to the target nucleic acidunder more favorable in solution kinetics before adjusting theconditions to permit hybridization of the immobilized probe bindingregion to the immobilized probe. The target binding and immobilizedprobe binding regions may be contained within the same oligonucleotide,directly adjoining each other or separated by one or more optionallymodified nucleotides, or these regions may be joined to each other bymeans of a non-nucleotide linker.

By “immobilized probe” is meant an oligonucleotide for joining a captureprobe to an immobilized support. The immobilized probe is joined eitherdirectly or indirectly to the solid support by a linkage or interactionwhich remains stable under the conditions employed to hybridize thecapture probe to the target nucleic acid and to the immobilized probe,whether those conditions are the same or different. The immobilizedprobe facilitates separation of the bound target nucleic acid fromunbound materials in a sample.

By “separating,” “purifying” or “purified” is meant that one or morecomponents of a sample contained in a sample-holding vessel are or havebeen physically removed from, or diluted in the presence of, one or moreother sample components present in the vessel. Sample components whichmay be removed or diluted during a separating or purifying step includeproteins, carbohydrates, lipids, inhibitors, non-target nucleic acidsand unbound probe. With target capture procedures, target nucleic acidsbound to immobilized capture probes are preferably retained in thesample during the separating or purifying step.

By “helper probe” or “helper oligonucleotide” is meant anoligonucleotide designed to hybridize to a target nucleic acid at adifferent locus than that of a hybridization assay probe, thereby eitherincreasing the rate of hybridization of the probe to the target nucleicacid, increasing the melting temperature (T_(m)) of the probe:targethybrid, or both.

By “phylogenetically closely related” is meant that the organisms areclosely related to each other in an evolutionary sense and thereforewould have a higher total nucleic acid sequence homology than organismsthat are more distantly related. Organisms occupying adjacent and nextto adjacent positions on the phylogenetic tree are closely related.Organisms occupying positions farther away than adjacent or next toadjacent positions on the phylogenetic tree will still be closelyrelated if they have significant total nucleic acid sequence homology.

By “genus-specific” is meant that the referred to probe is capable ofpreferentially hybridizing under stringent hybridization assayconditions to a target nucleic acid sequence present in nucleic acidderived from organisms belonging to at least two species of the genusCryptosporidium.

By “species-specific” is meant that the referred to probe is capable ofpreferentially hybridizing under stringent hybridization assayconditions to a target nucleic acid sequence present in nucleic acidderived from organisms belonging to the species Cryptosporidium parvum.

B. Hybridization Conditions and Probe/Primer Design

Hybridization reaction conditions, most importantly the temperature ofhybridization and the concentration of salt in the hybridizationsolution, can be selected to allow the hybridization assay probes oramplification primers of the present invention to preferentiallyhybridize to nucleic acids having a Cryptosporidium target nucleicsequence, and not to other non-target nucleic acids suspected of beingpresent in a test sample. At decreased salt concentrations and/orincreased temperatures (conditions of increased stringency) the extentof nucleic acid hybridization decreases as hydrogen bonding betweenpaired nucleotide bases in the double-stranded hybrid molecule isdisrupted. This process is known as “melting.”

Generally speaking, the most stable hybrids are those having the largestnumber of contiguous, perfectly matched (i.e., hydrogen-bonded)nucleotide base pairs. Such hybrids would usually be expected to be thelast to melt as the stringency of the hybridization conditionsincreases. However, a double-stranded nucleic acid region containing oneor more mismatched, “non-canonical,” or imperfect base pairs (resultingin weaker or non-existent base pairing at that position in thenucleotide sequence of a nucleic acid) may still be sufficiently stableunder conditions of relatively high stringency to allow the nucleic acidhybrid to be formed and detected in a hybridization assay withoutcross-reacting with other, non-selected nucleic acids which may bepresent in a test sample.

Hence, depending on the degree of similarity between the nucleotidesequences of the target nucleic acid and those of non-target nucleicacids belonging to phylogenetically distinct, but closely-relatedorganisms on one hand, and the degree of complementarity between thenucleotide sequences of a particular hybridization assay probe oramplification primer and those of the target and non-target nucleicacids on the other, one or more mismatches will not necessarily defeatthe ability of an oligonucleotide contained in the probe or primer tohybridize to the target nucleic acid and not to non-target nucleicacids.

The hybridization assay probes of the present invention were chosen,selected, and/or designed to maximize the difference between the meltingtemperatures of the probe:target hybrid (T_(m), defined as thetemperature at which half of the potentially double-stranded moleculesin a given reaction mixture are in a single-stranded, denatured state)and the T_(m) of a mismatched hybrid formed between the probe and rRNAor rDNA of the phylogenetically most closely-related organisms expectedto be present in the test sample, but not sought to be detected. Whilethe unlabeled amplification primers and helper probes need not have suchan extremely high degree of specificity as the hybridization assay probeto be useful in the present invention, they are designed in a similarmanner to preferentially hybridize to one or more target nucleic acidsover other nucleic acids under specified amplification or hybridizationassay conditions.

To facilitate the identification of nucleic acid sequences to be used inthe design of probes, nucleotide sequences from different organisms werefirst aligned to maximize homology. The nucleotide sequences used forthis comparison were obtained from the GenBank database and had thefollowing associated accession numbers: Cryptosporidium parvum(Accession Nos. L16996, L16997, L25642, AF040725 and AF015772),Cryptosporidium muris (Accession No. L19069), Cryptosporidium baileyi(Accession No. L19068), Cryptosporidium wrairi (Accession No. U11440),Escherichia coli (Accession No. Z83204), Cyclospora cayetanensis(Accession No. AF111183), Sarcocystis hominis (Accession No. AF006470),Entamoeba histolytica (Accession No. X64142) and Eimeria praecox(Accession No. U67120).

Within the rRNA molecule there is a close relationship between secondarystructure (caused in part by intra-molecular hydrogen bonding) andfunction. This fact imposes restrictions on evolutionary changes in theprimary nucleotide sequence causing the secondary structure to bemaintained. For example, if a base is changed in one “strand” of adouble helix (due to intra-molecular hydrogen bonding, both “strands”are part of the same rRNA molecule), a compensating substitution usuallyoccurs in the primary sequence of the other “strand” in order topreserve complementarity (this is referred to as co-variance), and thusthe necessary secondary structure. This allows two very different rRNAsequences to be aligned based both on the conserved primary sequence andalso on the conserved secondary structure elements. Potential targetsequences for the hybridization assay probes described herein wereidentified by noting variations in the homology of the alignedsequences.

The sequence evolution at each of the variable regions is mostlydivergent. Because of the divergence, corresponding rRNA variableregions of more distant phylogenetic relatives of Cryptosporidium showgreater differences from Cryptosporidium rRNA than do the rRNAs ofphylogenetically closer relatives. Similarly, corresponding rRNAvariable regions of more distant phylogenetic relatives ofCryptosporidium parvum show greater differences from Cryptosporidiumparvum rRNA than do the rRNAs of phylogenetically closer relatives.Sufficient variation between Cryptosporidium (i.e., Cryptosporidiumparvum, Cryptosporidium muris, Cryptosporidium baileyi andCryptosporidium wrairi) and Escherichia coli, Cyclospora cayetanensis,Sarcocystis hominis, Entamoeba histolytica and Eimeria praecox wasobserved to identify preferred target sites and design hybridizationassay probes useful for distinguishing Cryptosporidium organisms overnon-Cryptosporidium organisms. Likewise, sufficient variation betweenCryptosporidium parvum and Cryptosporidium muris, Cryptosporidiumbaileyi and Cryptosporidium wrairi was observed to identify preferredtarget sites and design hybridization assay probes useful fordistinguishing Cryptosporidium parvum organisms over non-Cryptosporidiumparvum organisms, especially the noted Cryptosporidium species.

We have identified sequences which vary between Cryptosporidium parvumand other Cryptosporidium species, and between members of the genusCryptosporidium and other organisms, by comparative analysis of rRNAsequences published in the GenBank sequence database and, in the case ofCryptosporidium parvum, further determined and confirmed in thelaboratory. Computers and computer programs which may be used or adaptedfor the purposes herein disclosed are commercially available. Weobserved sufficient similarity between Cryptosporidium parvum,Cryptosporidium muris, Cryptosporidium baileyi and Cryptosporidiumwrairi to design the present Cryptosporidium probes. We also observedsufficient variation between these same organisms to design the presentCryptosporidium parvum probes.

Merely identifying putatively unique potential target nucleotidesequences does not guarantee that a functionally genus-specific orspecies-specific hybridization assay probe may be made to hybridize toCryptosporidium or Cryptosporidium parvum rRNA or rDNA comprising thatsequence. Various other factors will determine the suitability of anucleic acid locus as a target site for genus-specific orspecies-specific probes. Because the extent and specificity ofhybridization reactions such as those described herein are affected by anumber of factors, manipulation of one or more of those factors willdetermine the exact sensitivity and specificity of a particularoligonucleotide, whether perfectly complementary to its target or not.The importance and effect of various assay conditions are known to thoseskilled in the art and are disclosed by Hogan, “Nucleic Acid Probes forDetection and/or Quantitation of Non-Viral Organisms,” U.S. Pat. No.5,840,488; Hogan et al., “Nucleic Acid Probes to Mycobacteriumgordonae,” U.S. Pat. No. 5,216,143; and Kohne, “Method for Detection,Identification and Quantitation of Non-Viral Organisms,” U.S Pat. No.4,851,330.

The desired temperature of hybridization and the hybridization solutioncomposition (such as salt concentration, detergents and other solutes)can also greatly affect the stability of double-stranded hybrids.Conditions such as ionic strength and the temperature at which a probewill be allowed to hybridize to a target must be taken into account inconstructing a genus-specific or species-specific probe. The thermalstability of hybrid nucleic acids generally increases with the ionicstrength of the reaction mixture. On the other hand, chemical reagentswhich disrupt hydrogen bonds, such as formamide, urea, dimethylsulfoxide and alcohols, can greatly reduce the thermal stability of thehybrids.

To maximize the specificity of a probe for its target, the subjectprobes of the present invention were designed to hybridize to theirtargets under conditions of high stringency. Under such conditions onlysingle nucleic acid strands having a high degree of complementarity willhybridize to each other. Single nucleic acid strands without such a highdegree of complementarity will not form hybrids. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity which should exist between two nucleic acid strands inorder to form a hybrid. Stringency is chosen to maximize the differencein stability between the hybrid formed between the probe and the targetnucleic acid and potential hybrids between the probe and any non-targetnucleic acids present in a test sample.

Proper specificity may be achieved by minimizing the length of thehybridization assay probe having perfect complementarity to sequences ofnon-target organisms, by avoiding G and C rich regions ofcomplementarity to non-target nucleic acids, and by constructing theprobe to contain as many destabilizing mismatches to non-targetsequences as possible. Whether a probe is appropriate for detecting onlya specific type of organism depends largely on the thermal stabilitydifference between probe:target hybrids versus probe:non-target hybrids.In designing probes, the differences in these T_(m) values should be aslarge as possible (preferably 2-5° C. or more). Manipulation of theT_(m) can be accomplished by changes to probe length and probecomposition (e.g., GC content versus AT content).

In general, the optimal hybridization temperature for oligonucleotideprobes is approximately 5° C. below the melting temperature for a givenduplex. Incubation at temperatures below the optimum temperature mayallow mismatched base sequences to hybridize and can therefore decreasespecificity. The longer the probe, the more hydrogen bonding betweenbase pairs and, in general, the higher the T_(m). Increasing thepercentage of G and C also increases the T_(m) because G-C base pairsexhibit additional hydrogen bonding and therefore greater thermalstability than A-T base pairs. Such considerations are known in the art.(See, e.g., J. SAMBROOK ET AL., MOLECULAR CLONING: A LABORATORY MANUAL,ch. 11 (2d ed. 1989).)

A preferred method to determine T_(m) measures hybridization using thewell known Hybridization Protection Assay (HPA) disclosed by Arnold etal., “Homogenous Protection Assay,” U.S. Pat. No. 5,283,174. The T_(m)can be measured using HPA in the following manner. Probe molecules arelabeled with an acridinium ester and permitted to form probe:targethybrids in a lithium succinate buffer (0.1 M lithium succinate buffer,pH 4.7, 20 mM EDTA, 15 mM aldrithiol-2, 1.2 M LiCl, 3% (v/v) ethanolabsolute, 2% (w/v) lithium lauryl sulfate) using an excess amount oftarget. Aliquots of the solution containing the probe:target hybrids arethen diluted in the lithium succinate buffered solution and incubatedfor five minutes at various temperatures starting below that of theanticipated T_(m) (typically 55° C.) and increasing in 2-5° C.increments. This solution is then diluted with a mild alkaline boratebuffer (600 mM boric acid, 240 mM NaOH, 1% (v/v) TRITON® X-100, pH 8.5)and incubated at an equal or lower temperature (for example 50° C.) forten minutes.

Under these conditions the acridinium ester attached to thesingle-stranded probe is hydrolyzed, while the acridinium ester attachedto hybridized probe is relatively protected from hydrolysis. Thus, theamount of acridinium ester remaining after hydrolysis treatment isproportional to the number of hybrid molecules. The remaining acridiniumester can be measured by monitoring the chemiluminescence produced fromthe remaining acridinium ester by adding hydrogen peroxide and alkali tothe solution. Chemiluminescence can be measured in a luminometer, suchas a LEADER® 450i luminometer (Gen-Probe Incorporated, San Diego, Calif;Cat. No. 3200i). The resulting data is plotted as percent of maximumsignal (usually from the lowest temperature) versus temperature. TheT_(m) is defined as the temperature at which 50% of the maximum signalremains. In addition to the method above, T_(m) may be determined byisotopic methods known to those skilled in the art (see, e.g., U.S. Pat.No. 5,840,488).

It should be noted that the T_(m) for a given hybrid varies depending onthe nature of the hybridization solution used. Factors such as the saltconcentration, detergents, and other solutes can affect hybrid stabilityduring thermal denaturation (see, e.g., SAMBROOK ET AL., supra, ch. 11).Conditions such as ionic strength and the temperature at which a probewill be allowed to hybridize to target should be taken into account inprobe construction. (The thermal stability of a hybrid nucleic acidincreases with the ionic strength of the reaction mixture.) On the otherhand, chemical reagents that disrupt hydrogen bonds, such as formamide,urea, dimethyl sulfoxide and alcohols, can greatly reduce hybrid thermalstability.

To ensure specificity of a hybridization assay probe for its target, itis preferable to design probes which hybridize only to target nucleicacid under conditions of high stringency. Only highly complementarysequences will form hybrids under conditions of high stringency.Accordingly, the stringency of the assay conditions determines theamount of complementarity needed between two sequences in order for astable hybrid to form. Stringency should be chosen to maximize thedifference in stability between the probe:target hybrid and potentialprobe:non-target hybrids.

Examples of specific stringent hybridization conditions are provided inthe Examples section infra. Of course, alternative stringenthybridization conditions could be determined by those of ordinary skillin the art based on the present disclosure. (See, e.g., SAMBROOK ET AL.,supra, ch. 11.)

The length of the target nucleic acid sequence region and, accordingly,the length of the probe sequence can also be important. In some cases,there may be several sequences from a particular region, varying inlocation and length, which may be used to design probes with the desiredhybridization characteristics. In other cases, one probe may besignificantly better with regard to specificity than another whichdiffers from it merely by a single base. While it is possible fornucleic acids that are not perfectly complementary to hybridize, thelongest stretch of perfectly complementary bases, as well as the basecompositions, will generally determine hybrid stability.

Regions of rRNA known to form strong internal structures inhibitory tohybridization are less preferred target regions, especially in assayswhere helper probes described infra are not used. Likewise, probes withextensive self-complementarity are generally to be avoided, withspecific exceptions being discussed below. If a strand is wholly orpartially involved in an intramolecular or intermolecular hybrid, itwill be less able to participate in the formation of a newintermolecular probe:target hybrid without a change in the reactionconditions. Ribosomal RNA molecules are known to form very stableintramolecular helices and secondary structures by hydrogen bonding. Bydesigning a probe to a region of the target nucleic acid which remainssubstantially single-stranded under hybridization conditions, the rateand extent of hybridization between probe and target may be increased.

A genomic ribosomal nucleic acid (rDNA) target occurs naturally in adouble-stranded form, as does the product of the polymerase chainreaction (PCR). These double-stranded targets are naturally inhibitoryto hybridization with a probe and require denaturation prior tohybridization. Appropriate denaturation and hybridization conditions areknown in the art (see, e.g., Southern, E. M., J. Mol. Biol., 98:503(1975)).

A number of formulae are available which will provide an estimate of themelting temperature for perfectly matched oligonucleotides to theirtarget nucleic acids. One such formula is the following:T_(m)=81.5+16.6(log₁₀[Na⁺])+0.41(fraction G+C)−(600/N)(where N=the length of the oligonucleotide in number of nucleotides)provides a good estimate of the T_(m) for oligonucleotides between 14and 60 to 70 nucleotides in length. From such calculations, subsequentempirical verification or “fine tuning” of the T_(m) may be made usingscreening techniques well known in the art. For further information onhybridization and oligonucleotide probes reference may be made toSAMBROOK ET AL., supra, ch. 11. This reference, among others well knownin the art, also provides estimates of the effect of mismatches on theT_(m) of a hybrid. Thus, from the known nucleotide sequence of a givenregion of the ribosomal RNA (or rDNA) of two or more organisms,oligonucleotides may be designed which will distinguish these organismsfrom one another.

C. Nucleic Acid Amplification

Preferably, the amplification primers of the present invention areoligodeoxynucleotides and are sufficiently long to be used as asubstrate for the synthesis of extension products by a nucleic acidpolymerase. Optimal primer length should take into account severalfactors, including the temperature of reaction, the structure and basecomposition of the primer, and how the primer is to be used. Forexample, for optimal specificity the oligonucleotide primer generallyshould be at least 12 bases in length, depending on the complexity ofthe target nucleic acid sequence. If such specificity is not essential,shorter primers may be used. In such a case, it may be desirable tocarry out the reaction at cooler temperatures in order to form stablehybrid complexes with the template nucleic acid.

Useful guidelines for designing amplification primers and hybridizationassay probes with desired characteristics are described infra in thesection entitled “Preparation of Oligonucleotides.” Optimal sites foramplifying and probing contain at least two, and preferably three,conserved regions of Cryptosporidium or Cryptosporidium parvum nucleicacid. These regions are about 15 to 350 bases in length, and preferablybetween about 15 and 150 bases in length.

The degree of amplification observed with a set of amplification primers(primers and/or promoter-primers) depends on several factors, includingthe ability of the primers to hybridize to their specific targetsequences and their ability to be extended or copied enzymatically.While amplification primers of different lengths and base compositionsmay be used, amplification primers preferred in this invention havetarget binding regions of 18 to 40 bases with a predicted T_(m) totarget of about 42° C.

Parameters affecting probe hybridization, such as T_(m), complementarityand secondary structure of the target sequence, also affectamplification primer hybridization and therefore performance of theamplification primers. The degree of non-specific extension(primer-dimer or non-target copying) can also affect amplificationefficiency. Thus, amplification primers are selected to have lowself-complementarity or cross-complementarity, particularly at the 3′ends of their sequences. Notwithstanding, we note that the “signalprimers” described infra could be modified to include regions ofself-complementarity, thereby transforming them into “Molecular Torch”or “Molecular Beacon” signal primers, such as these terms are definedbelow. Lengthy homopolymer runs and high GC content are avoided toreduce spurious primer extension. Computer programs are available to aidin this aspect of the design, including Oligo Tech® analysis softwarewhich is available from Oligo Therapeutics, Inc. and can be accessed onthe World Wide Web at www.oligosetc.com/OligoTech.html using a hypertexttransfer protocol (http) in the URL.

A nucleic acid polymerase used in conjunction with the amplificationprimers of the present invention refers to a chemical, physical orbiological agent which incorporates either ribonucleotides ordeoxyribonucleotides, or both, into a nucleic acid polymer, or strand,in a template-dependent manner. Examples of nucleic acid polymerasesinclude DNA-directed DNA polymerases, RNA-directed DNA polymerases, andRNA-directed RNA polymerases. DNA polymerases bring about nucleic acidsynthesis in a template-dependent manner and in a 5′ to 3′ direction.Because of the typical anti-parallel orientation of the two strands in adouble-stranded nucleic acid, this direction is from a 3′ region on thetemplate to a 5′ region on the template. Examples of DNA-directed DNApolymerases include E. coli DNA polymerase I, the thermostable DNApolymerase from Thermus aquaticus (Taq), and the large fragment of DNApolymerase I from Bacillus stearothermophilus (Bst). Examples of RNAdirected DNA polymerases include various retroviral reversetranscriptases, such as Moloney murine leukemia virus (MMLV) reversetranscriptase or avian myeloblastosis virus (AMV) reverse transcriptase.

During most nucleic acid amplification reactions, a nucleic acidpolymerase adds nucleotide residues to the 3′ end of the primer usingthe target nucleic acid as a template, thus synthesizing a secondnucleic acid strand having a nucleotide sequence partially or completelycomplementary to a region of the target nucleic acid. In many nucleicacid amplification reactions, the two strands comprising the resultingdouble-stranded structure must be separated by chemical or physicalmeans in order to allow the amplification reaction to proceed.Alternatively, the newly-synthesized template strand may be madeavailable for hybridization with a second primer or promoter-primer byother means, such as through strand displacement or the use of anucleolytic enzyme which digests part or all of the original targetstrand. In this way the process may be repeated through a number ofcycles, resulting in a large increase in the number of nucleic acidmolecules having the target nucleotide sequence.

Either the first or second amplification primer, or both, may be apromoter-primer. (In some applications, the amplification primers mayonly consist of promoter-primers which are complementary to the sensestrand, as disclosed by Kacian et al., “Nucleic Acid SequenceAmplification Method, Composition and Kit,” U.S. Pat. No. 5,554,516.) Apromoter-primer usually contains an oligonucleotide that is notcomplementary to a nucleotide sequence present in the target nucleicacid molecule or primer extension product(s) (see Kacian et al.,“Nucleic Acid Sequence Amplification Methods,” U.S. Pat. No. 5,399,491,for a description of such oligonucleotides). These non-complementarysequences may be located 5′ to the complementary sequences on theamplification primer and may provide a locus for initiation of RNAsynthesis when made double-stranded through the action of a nucleic acidpolymerase. The promoter thus provided may allow for the in vitrotranscription of multiple RNA copies of the target nucleic acidsequence. It will be appreciated that when reference is made to a primerin this specification, such reference is intended to include the primeraspect of a promoter-primer as well, unless the context of the referenceclearly indicates otherwise.

In some amplification systems (see, e.g., the amplification methodsdisclosed by Dattagupta et al, “Isothermal Strand Displacement NucleicAcid Amplification,” U.S. Pat. No. 6,087,133), the amplification primersmay contain 5′ non-complementary nucleotides which assist in stranddisplacement. Furthermore, when used in conjunction with a nucleic acidpolymerase having 5′ exonuclease activity, the amplification primers mayhave modifications at their 5′ end to prevent enzymatic digestion.Alternatively, the nucleic acid polymerase may be modified to remove the5′ exonuclease activity, such as by treatment with a protease thatgenerates an active polymerase fragment with no such nuclease activity.In such a case the primers need not be modified at their 5′ ends.

1. Preparation of Oligonucleotides

The oligonucleotide primers and probes of the present invention can bereadily prepared by methods known in the art. Preferably, theoligonucleotides are synthesized using solid phase methods. For example,Caruthers describes using standard phosphoramidite solid-phase chemistryto join nucleotides by phosphodiester linkages. (See Caruthers, M. H.,et al., Methods Enzymol., 154:287 (1987).) Automated solid-phasechemical synthesis using cyanoethyl phosphoramidite precursors has beendescribed by Barone. (See Barone, A. D., et al., Nucleic Acids Res.,12(10):4051 (1984).) Likewise, Batt, “Method and Reagent forSulfurization of Organophosphorous Compounds,” U.S. Pat. No. 5,449,769,discloses a procedure for synthesizing oligonucleotides containingphosphorothioate linkages. In addition, Riley et al., “Process for thePurification of Oligomers,” U.S. Pat. No. 5,811,538, disclose thesynthesis of oligonucleotides having different linkages includingmethylphosphonate linkages. Moreover, methods for the organic synthesisof oligonucleotides are known to those of skill in the art and aredescribed in, for example, SAMBROOK ET AL., supra, ch. 10.

Following synthesis and purification of a particular oligonucleotide,several different procedures may be utilized to purify and control thequality of the oligonucleotide. Suitable procedures includepolyacrylamide gel electrophoresis or high pressure liquidchromatography. Both of these procedures are well known to those skilledin the art.

All of the oligonucleotides of the present invention, whetherhybridization assay probes, amplification primers or helper probes, maybe modified with chemical groups to enhance their performance or tofacilitate the characterization of amplification products.

For example, backbone-modified oligonucleotides such as those havingphosphorothioate, methylphosphonate, 2′-O-alkyl or peptide groups whichrender the oligonucleotides resistant to the nucleolytic activity ofcertain polymerases or to nuclease enzymes may allow the use of suchenzymes in an amplification or other reaction. Another example of amodification involves using non-nucleotide linkers (see Arnold et al.,“Non-Nucleotide Linking Reagents for Nucleotide Probes,” U.S. Pat. No.6,031,091.) incorporated between nucleotides in the nucleic acid chainof a probe or primer, and which do not prevent hybridization of a probeor hybridization and elongation of a primer. The oligonucleotides of thepresent invention may also contain mixtures of the desired modified andnatural nucleotides.

The 3′ end of an amplification primer may be modified or blocked toprevent or inhibit initiation of DNA synthesis, as disclosed by Kacianet al. in U.S. Pat. No. 5,554,516. The 3′ end of the primer can bemodified in a variety of ways well known in the art. By way of example,appropriate modifications to a primer can include the addition ofribonucleotides, 3′ deoxynucleotide residues (e.g., cordycepin),2′,3′-dideoxynucleotide residues, modified nucleotides such asphosphorothioates, and non-nucleotide linkages such as those disclosedby Arnold et al. in U.S. Pat. No. 6,031,091 or alkane-diol modifications(see Wilk et al., Nucleic Acids Res., 18:2065 (1990)), or themodification may simply consist of a region 3′ to the priming sequencethat is non-complementary to the target nucleic acid sequence.Additionally, a mixture of different 3′ blocked primers or of 3′ blockedand unblocked primers may increase the efficiency of nucleic acidamplification, as described therein.

As disclosed above, the 5′ end of primers may be modified to beresistant to the 5′-exonuclease activity present in some nucleic acidpolymerases. Such modifications can be carried out by adding anon-nucleotide group to the terminal 5′ nucleotide of the primer usingtechniques such as those disclosed by Arnold et al. in U.S. Pat.No.6,031,091.

Once synthesized, a selected oligonucleotide may be labeled by any ofseveral well known methods (see, e.g., SAMBROOK, supra, ch. 10). Usefullabels include radioisotopes as well as non-radioactive reportinggroups. Isotopic labels include ³H, ³⁵S, ³²P, ¹²⁵I, ⁵⁷Co and ¹⁴C.Isotopic labels can be introduced into the oligonucleotide by techniquesknown in the art such as nick translation, end labeling, second strandsynthesis, the use of reverse transcription, and by chemical methods.When using radiolabeled probes, hybridization can be detected byautoradiography, scintillation counting or gamma counting. The detectionmethod selected will depend upon the particular radioisotope used forlabeling.

Non-isotopic materials can also be used for labeling and may beintroduced internally into the nucleic acid sequence or at the end ofthe nucleic acid sequence. Modified nucleotides may be incorporatedenzymatically or chemically. Chemical modifications of the probe may beperformed during or after synthesis of the probe, for example, throughthe use of non-nucleotide linker groups as disclosed by Arnold et al. inU.S. Pat. No. 6,031,091. Non-isotopic labels include fluorescentmolecules (individual labels or combinations of labels such as thefluorescence resonance energy transfer (FRET) pairs disclosed by Tyagiet al., “Detectably Labeled Dual Conformation Oligonucleotide Probes,Assays and Kits,” U.S. Pat. No. 5,925,517), chemiluminescent molecules,enzymes, cofactors, enzyme substrates, haptens or other ligands.

With the hybridization assay probes of the present invention, the probesare preferably labeled by means of a non-nucleotide linker with anacridinium ester. Acridinium ester labeling may be performed asdisclosed by Arnold et al., “Acridinium Ester Labelling and Purificationof Nucleotide Probes,” U.S. Pat. No. 5,185,439.

2. Amplification of Cryptosporidium Ribosomal Nucleic Acid

The amplification primers of the present invention are directed toregions of 18S ribosomal nucleic acid derived from Cryptosporidium orCryptosporidium parvum organisms. These amplification primers may flank,overlap or be contained within at least one of the target nucleic acidsequences of a hybridization assay probe (or its complement) used todetect the presence of Cryptosporidium or Cryptosporidium parvumorganisms in a nucleic acid amplification assay. As indicated above, theamplification primers may also include non-complementary bases at their5′ ends comprising a promoter sequence able to bind an RNA polymeraseand direct RNA transcription using the target nucleic acid as atemplate. A T7 promoter sequence, such as SEQ ID NO:69, may be used.

Amplification primers of the present invention are capable of amplifyinga target nucleic acid sequence present in nucleic acid derived fromCryptosporidium organisms under amplification conditions. Theseamplification primers comprise an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67 or SEQ ID NO:68.

Alternatively, amplification primers of the present invention comprisean oligonucleotide which, when contacted with a nucleic acid polymeraseunder amplification conditions, will bind to or cause extension througha nucleic acid region having a base sequence of SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ IDNO:66, SEQ ID NO:67 or SEQ ID NO:68.

In one preferred embodiment, a set of at least two amplification primersfor amplifying Cryptosporidium nucleic acid is provided which includes:(i) a first amplification primer comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:45, SEQ IDNO:51, SEQ ID NO:57 or SEQ ID NO:63; and (ii) a second amplificationprimer comprising an oligonucleotide having or substantiallycorresponding to the base sequence of SEQ ID NO:47, SEQ ID NO:53, SEQ IDNO:59 or SEQ ID NO:65. Preferably, the first amplification primercomprises an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:45, and the second amplification primercomprises an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:59.

In another preferred embodiment, a set of at least two amplificationprimers for amplifying Cryptosporidium nucleic acid is provided whichincludes: (i) a first amplification primer comprising an oligonucleotidehaving or substantially corresponding to the base sequence of SEQ IDNO:45, SEQ ID NO:51, SEQ ID NO:57 or SEQ ID NO:63; and (ii) a secondamplification primer comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:48, SEQ IDNO:54, SEQ ID NO:60 or SEQ ID NO:66. Preferably, the first amplificationprimer comprises an oligonucleotide having or substantiallycorresponding to the base sequence of SEQ ID NO:45, and the secondamplification primer comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:60.

In yet another preferred embodiment, a set of at least two amplificationprimers for amplifying Cryptosporidium nucleic acid is provided whichincludes: (i) a first amplification primer comprising an oligonucleotidehaving or substantially corresponding to the base sequence of SEQ IDNO:46, SEQ ID NO:52, SEQ ID NO:58 or SEQ ID NO:64; and (ii) a secondamplification primer comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:48, SEQ IDNO:54, SEQ ID NO:60 or SEQ ID NO:66. Preferably, the first amplificationprimer comprises an oligonucleotide having or substantiallycorresponding to the base sequence of SEQ ID NO:46, and the secondamplification primer comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:60.

In still another preferred embodiment, a set of at least twoamplification primers for amplifying Cryptosporidium nucleic acid isprovided which includes: (i) a first amplification primer comprising anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:46, SEQ ID NO:52, SEQ ID NO:58 or SEQ ID NO:64;and (ii) a second amplification primer comprising an oligonucleotidehaving or substantially corresponding to the base sequence of SEQ IDNO:47, SEQ ID NO:53, SEQ ID NO:59 or SEQ ID NO:65. Preferably, the firstamplification primer comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:46, andthe second amplification primer comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:59.

In a further preferred embodiment, a set of at least two amplificationprimers for amplifying Cryptosporidium nucleic acid is provided whichincludes: (i) a first amplification primer comprising an oligonucleotidehaving or substantially corresponding to the base sequence of SEQ IDNO:49, SEQ ID NO:55, SEQ ID NO:61 or SEQ ID NO:67; and (ii) a secondamplification primer comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:50, SEQ IDNO:56, SEQ ID NO:62 or SEQ ID NO:68. Preferably, the first amplificationprimer comprises an oligonucleotide having or substantiallycorresponding to the base sequence of SEQ ID NO:49, and the secondamplification primer comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:62.

Amplification primers of the present invention may have modifications,such as blocked 3′ and/or 5′ termini (as discussed above) or sequenceadditions including, but not limited to, a specific nucleotide sequencerecognized by an RNA polymerase (e.g., the promoter sequence for T7, T3or SP6 RNA polymerase), a sequence which enhances initiation orelongation of RNA transcription by an RNA polymerase, or a sequencewhich may provide for intra-molecular base pairing and encourage theformation of secondary or tertiary nucleic acid structures.

Amplification primers are used in a nucleic acid amplificationprocedure, such as the polymerase chain reaction (PCR),transcription-mediated amplification (TMA), nucleic acid sequence-basedamplification (NASBA), self-sustained sequence replication (3SR), ligasechain reaction (LCR), strand displacement amplification (SDA), andLoop-Mediated Isothermal Amplification (LAMP), each of which is wellknown in the art. See, e.g., Mullis, “Process for Amplifying NucleicAcid Sequences,” U.S. Pat. No. 4,683,202; Erlich et al., “Kits forAmplifying and Detecting Nucleic Acid Sequences,” U.S. Pat. No.6,197,563; Walker et al., Nucleic Acids Res., 20:1691-1696 (1992); Fahyet al., “Self-sustained Sequence Replication (3SR): An IsothermalTranscription-Based Amplification System Alternative to PCR,” PCRMethods and Applications, 1:25-33 (1991); Kacian et al., U.S. Pat. No.5,399,491; Kacian et al., “Nucleic Acid Sequence Amplification Methods,”U.S. Pat. No. 5,480,784; Davey et al., “Nucleic Acid AmplificationProcess,” U.S. Pat. No. 5,554,517; Birkenmeyer et al., “Amplification ofTarget Nucleic Acids Using Gap Filling Ligase Chain Reaction,” U.S. Pat.No. 5,427,930; Marshall et al., “Amplification of RNA Sequences Usingthe Ligase Chain Reaction,” U.S. Pat. No. 5,686,272; Walker, “StrandDisplacement Amplification,” U.S. Pat. No. 5,712,124; Notomi et al.,“Process for Synthesizing Nucleic Acid,” European Patent Application No.1 020 534 A1; Dattagupta et al., “Isothermal Strand DisplacementAmplification,” U.S. Pat. No. 6,214,587; and HELEN H. LEE ET AL.,NUCLEIC ACID AMPLIFICATION TECHNOLOGIES: APPLICATION TO DISEASEDIAGNOSIS (1997). Any other amplification procedure which meets thedefinition of “nucleic acid amplification” supra is also contemplated bythe inventors.

Amplification primers of the present invention are preferably unlabeledbut may include one or more reporter groups to facilitate detection of atarget nucleic acid in combination with or exclusive of a hybridizationassay probe. A wide variety of methods are available to detect anamplified target sequence. For example, the nucleotide substrates or theprimers can include a detectable label which is incorporated into newlysynthesized DNA. The resulting labeled amplification product is thengenerally separated from the unused labeled nucleotides or primers andthe label is detected in the separated product fraction. (See, e.g., Wu,“Detection of Amplified Nucleic Acid Using Secondary CaptureOligonucleotides and Test Kit,” U.S. Pat. No. 5,387,510.)

A separation step is not required, however, if the primer is modifiedby, for example, linking it to two dyes which form a donor/acceptor dyepair. The modified primer can be designed so that the fluorescence ofone dye pair member remains quenched by the other dye pair member, solong as the primer does not hybridize to target nucleic acid, therebyphysically separating the two dyes. Moreover, the primer can be furthermodified to include a restriction endonuclease recognition sitepositioned between the two dyes so that when a hybrid is formed betweenthe modified primer and target nucleic acid, the restrictionendonuclease recognition site is rendered double-stranded and availablefor cleavage or nicking by an appropriate restriction endonuclease.Cleavage or nicking of the hybrid then separates the two dyes, resultingin a change in fluorescence due to decreased quenching which can bedetected as an indication of the presence of the target organism ororganisms in the test sample. This type of modified primer, referred toas a “signal primer,” is disclosed by Nadeau et al., “Detection ofNucleic Acids by Fluorescence Quenching,” U.S. Pat. No. 6,054,279.

Substances which can serve as useful detectable labels are well known inthe art and include radioactive isotopes, fluorescent molecules,chemiluminescent molecules, chromophores, as well as ligands such asbiotin and haptens which, while not directly detectable, can be readilydetected by a reaction with labeled forms of their specific bindingpartners, e.g., avidin and antibodies, respectively.

Another approach is to detect the amplification product by hybridizationwith a detectably labeled oligonucleotide probe and measuring theresulting hybrids in any conventional manner. In particular, the productcan be assayed by hybridizing a chemiluminescent acridiniumester-labeled oligonucleotide probe to the target sequence, selectivelyhydrolyzing the acridinium ester present on unhybridized probe, andmeasuring the chemiluminescence produced from the remaining acridiniumester in a luminometer. (See, e.g., U.S. Pat. No. 5,283,174 and NORMANC. NELSON ET AL., NONISOTOPIC PROBING, BLOTTING, AND SEQUENCING, ch. 17(Larry J. Kricka ed., 2d ed. 1995).)

D. Hybridization Assay Probes to Cryptosporidium Ribosomal Nucleic Acid

This embodiment of the invention relates to novel hybridization assayprobes. Hybridization is the association of two single strands ofcomplementary nucleic acid to form a hydrogen bonded double strand. Anucleic acid sequence able to hybridize to a nucleic acid sequencesought to be detected (“target sequence”) can serve as a probe for thetarget sequence. Hybridization may occur between complementary nucleicacid strands, including DNA/DNA, DNA/RNA, and RNA/RNA. Two singlestrands of deoxyribo-(DNA) or ribo-(RNA) nucleic acid, formed fromnucleotides (including the bases adenine (A), cytosine (C), thymidine(T), guanine (G), uracil (U), inosine (I), and analogs thereof), mayhybridize to form a double-stranded structure in which the two strandsare held together by hydrogen bonds between pairs of complementarybases. Generally, A is hydrogen-bonded to T or U, while G ishydrogen-bonded to C. At any point along the hybridized strands,therefore, the classical base pairs AT or AU, TA or UA, GC, or CG may befound. Thus, when a first single strand of nucleic acid containssufficient contiguous complementary bases to a second, and those twostrands are brought together under conditions that will promote theirhybridization, double-stranded nucleic acid will result. Underappropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids may beformed.

The rate and extent of hybridization is influenced by a number offactors. For instance, it is implicit that if one of the two strands iswholly or partially involved in a hybrid, it will be less able toparticipate in the formation of a new hybrid. By designing a probe sothat a substantial portion of the sequence of interest issingle-stranded, the rate and extent of hybridization may be greatlyincreased. Also, if the target is an integrated genomic sequence it willnaturally occur in a double-stranded form, as is the case with a productof PCR. These double-stranded targets are naturally inhibitory tohybridization with a probe and require denaturation prior to thehybridization step. In addition, there can be intra-molecular andinter-molecular hybrids formed within a probe if there is sufficientself-complementarity. Regions of the nucleic acid which are known toform strong internal structures inhibitory to hybridization are lesspreferred. Examples of such structures include hairpin loops. Likewise,probes with extensive self-complementarity generally should be avoided.All these undesirable structures can be avoided through careful probedesign, and commercial computer programs are available to search forthese types of interactions, such as the Oligo Tech® analysis softwareavailable from Oligo Therapeutics, Inc.

In some applications, probes exhibiting at least some degree ofself-complementarity are desirable to facilitate detection ofprobe:target duplexes in a test sample without first requiring theremoval of unhybridized probe prior to detection. By way of example,structures referred to as “Molecular Torches” are designed to includedistinct regions of self-complementarity (coined “the target bindingdomain” and “the target closing domain”) which are connected by ajoining region and which hybridize to one another under predeterminedhybridization assay conditions. When exposed to denaturing conditions,the two complementary regions (which may be fully or partiallycomplementary) of the Molecular Torch melt, leaving the target bindingdomain available for hybridization to a target sequence when thepredetermined hybridization assay conditions are restored. MolecularTorches are designed so that the target binding domain favorshybridization to the target sequence over the target closing domain. Thetarget binding domain and the target closing domain of a Molecular Torchinclude interacting labels (e.g., luminescent/quencher) positioned sothat a different signal is produced when the Molecular Torch isself-hybridized than when the Molecular Torch is hybridized to a targetnucleic acid, thereby permitting detection of probe:target duplexes in atest sample in the presence of unhybridized probe having a viable labelassociated therewith. (Molecular Torches are disclosed by Becker et al.,“Molecular Torches,” U.S. application Ser. No. 09/346,551 andInternational Publication No. WO 00/01850, both of which enjoy commonownership herewith.)

Another example of a hybridization assay probe havingself-complementarity is a structure commonly referred to as a “MolecularBeacon.” Molecular Beacons include nucleic acid molecules having atarget complement sequence, an affinity pair (or nucleic acid arms)holding the probe in a closed conformation in the absence of a targetnucleic acid sequence, and a label pair that interacts when the probe isin a closed conformation. Hybridization of the target nucleic acid andthe target complement sequence separates the members of the affinitypair, thereby shifting the probe to an open confirmation. The shift tothe open confirmation is detectable due to reduced interaction of thelabel pair, which may be, for example, a fluorophore and a quencher(e.g., DABCYL and EDANS). (Molecular Beacons are disclosed by Tyagi etal. in U.S. Pat. No. 5,925,517.)

The rate at which a probe hybridizes to its target is one measure of thethermal stability of the target secondary structure in the probe region.The standard measurement of hybridization rate is the C_(o)t_(1/2),which is measured as moles of nucleotide per liter times seconds. Thus,it is the concentration of probe times the time at which 50% of maximalhybridization occurs at that concentration. This value is determined byhybridizing various amounts of probe to a constant amount of target fora fixed time. The C_(o)t_(1/2) is found graphically by standardprocedure. The probe:target hybrid melting temperature may be determinedby isotopic methods well-known to those skilled in the art. The meltingtemperature (T_(m)) for a given hybrid will vary depending on thehybridization solution being used.

Thus, in a first aspect, the invention features hybridization assayprobes able to distinguish Cryptosporidium nucleic acid fromnon-Cryptosporidium nucleic acid, by virtue of the ability of the probeto preferentially hybridize to Cryptosporidium nucleic acid understringent hybridization assay conditions. Specifically, theCryptosporidium probes contain an oligonucleotide having a base sequencethat is substantially complementary to a target sequence present innucleic acid derived from Cryptosporidium organisms. A Cryptosporidiumprobe of the present invention may detect less than all members of thegenus Cryptosporidium which may be present in a test sample and still becharacterized as a Cryptosporidium probe, provided the Cryptosporidiumprobe is capable of detecting the presence of at least two speciesbelonging to the Cryptosporidium genus under stringent hybridizationassay conditions.

In a related aspect, the invention describes hybridization assay probesable to distinguish Cryptosporidium parvum nucleic acid fromnon-Cryptosporidium parvum nucleic acid, by virtue of the ability of theprobe to preferentially hybridize to Cryptosporidium parvum nucleic acidunder stringent hybridization assay conditions. Specifically, theCryptosporidium parvum probes contain an oligonucleotide having a basesequence that is substantially complementary to a target sequencepresent in nucleic acid derived from Cryptosporidium parvum organisms. ACryptosporidium parvum probe of the present invention may detect lessthan all members of the species Cryptosporidium parvum which may bepresent in a test sample and still be characterized as a Cryptoporidiumparvum probe, provided the Cryptosporidium parvum probe is capable ofdetecting the presence of at least one strain belonging to theCryptosporidium parvum species under stringent hybridization assayconditions.

In the case of a hybridization assay, the length of the target nucleicacid sequence and, accordingly, the length of the probe sequence can beimportant. In some cases, there may be several sequences from aparticular region, varying in location and length, which will yieldprobes with the desired hybridization characteristics. In other cases,one sequence may have better hybridization characteristics than anotherthat differs merely by a single base. While it is possible for nucleicacids that are not perfectly complementary to hybridize, the longeststretch of perfectly homologous base sequence will normally primarilydetermine hybrid stability. While probes of different lengths and basecomposition may be used, the probes preferred in this invention haveoligonucleotides that are up to 100 bases in length, more preferablyfrom 12 to 50 bases in length, and even more preferably from 18 to 35bases in length.

The hybridization assay probes include a base sequence that issubstantially complementary to an 18S ribosomal RNA (rRNA), or theencoding DNA (rDNA), target sequence of Cryptosporidium orCryptosporidium parvum. Thus, the probes are able to stably hybridize toa target sequence derived from a Cryptosporidium or Cryptosporidiumparvum organism or organisms under stringent hybridization assayconditions. The hybridization assay probes may also have additionalbases outside of the targeted nucleic acid region which may or may notbe complementary to Cryptosporidium-derived or Cryptosporidiumparvum-derived nucleic acid but which are not complementary to nucleicacid derived from a non-target organism which may be present in the testsample.

Probes (and primers) of the present invention may also be designed toinclude a capture tail comprised of a base sequence (distinct from thebase sequence intended to hybridize to the target sequence) which canhybridize under predetermined hybridization conditions to asubstantially complementary base sequence present in an immobilizedoligonucleotide which is joined to a solid support. The immobilizedoligonucleotide is preferably joined to a magnetically charged particlewhich can be isolated in a reaction vessel during a purification stepafter a sufficient period of time has passed for probe to hybridize totarget nucleic acid. (An example of an instrument which can be used toperform such a purification step is disclosed by Acosta et al., “AssayWork Station,” U.S. Pat. No. 6,254,826.) The probe is preferablydesigned so that the melting temperature of the probe:target hybrid isgreater than the melting temperature of the probe:immobilizedoligonucleotide hybrid. In this way, different sets of hybridizationassay conditions can be employed to facilitate hybridization of theprobe to the target nucleic acid prior to hybridization of the probe tothe immobilized oligonucleotide, thereby maximizing the concentration offree probe and providing favorable liquid phase hybridization kinetics.This “two-step” target capture method is disclosed by Weisburg et al.,“Two Step Hybridization and Capture of a Polynucleotide,” U.S. Pat. No.6,110,678. Other target capture schemes which could be readily adaptedto the present invention are well known in the art and include, forexample, those disclosed by Ranki et al., “Detection of MicrobialNucleic Acids by a One-Step Sandwich Hybridization Test,” U.S. Pat. No.4,486,539, and Stabinsky, “Methods and Kits for Performing Nucleic AcidHybridization Assays,” U.S. Pat. No. 4,751,177.

For Cryptosporidium probes, the terms “target nucleic acid sequence,”“target nucleotide sequence,” “target sequence” and “target region” allrefer to a nucleic acid sequence present in Cryptosporidium rRNA orrDNA, or a sequence complementary thereto, which is not present in thenucleic acid of a closely related non-Cryptosporidium species. ForCryptosporidium parvum probes, the terms “target nucleic acid sequence,”“target nucleotide sequence,” “target sequence” and “target region” allrefer to a nucleic acid sequence present in Cryptosporidium parvum rRNAor rDNA, or a sequence complementary thereto, which is not present inthe nucleic of a closely related non-Cryptosporidium parvum species.Nucleic acids having nucleotide sequences complementary to a targetsequence may be generated by target amplification techniques such as thepolymerase chain reaction (PCR) or transcription-mediated amplification(TMA). (TMA is disclosed by, for example, Kacian et al. in U.S. Pat.Nos. 5,399,491, and Kacian et al, “Nucleic Acid Sequence AmplificationMethods,” U.S. Pat. No. 5,480,784.)

Organisms that might be expected to be present in aCryptosporidium-containing test sample include, for example, Escherichiacoli, Cyclospora cayetanensis, Sarcocystis hominis, Entamoebahistolytica and Eimeria praecox. This list of organisms is by no meansintended to be fully representative of the organisms that theCryptosporidium probes of the present invention can be used todistinguish over. In general, the Cryptosporidium probes of thisinvention can be used to distinguish Cryptosporidium nucleic acid fromany non-Cryptosporidium nucleic acid that does not stably hybridize withthe probe(s) under stringent hybridization conditions.

Organisms closely related to Cryptosporidium parvum includeCryptosporidium muris, Cryptosporidium baileyi and Cryptosporidiumwrairi, although this list is by no means intended to be fullyrepresentative of the organisms that the Cryptosporidium parvum probesof the present invention can be used to distinguish over. In general,the Cryptosporidium parvum probes of this invention can be used todistinguish Cryptosporidium parvum nucleic acid from anynon-Cryptosporidium parvum nucleic acid that does not stably hybridizewith the probe(s) under stringent hybridization conditions.

A Cryptosporidium probe of the present invention preferably comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. ACryptosporidium probe comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO: 1 or SEQID NO:2 preferably includes an acridinium ester label joined to theprobe by means of a non-nucleotide linker positioned between nucleotides11 and 12 (reading 5′ to 3′) of SEQ ID NO:1 or SEQ ID NO:2. And aCryptosporidium probe comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:3 or SEQID NO:4 preferably includes an acridinium ester label joined to theprobe by means of a non-nucleotide linker positioned between nucleotides11 and 12 (reading 5′ to 3′) of SEQ ID NO:3 or SEQ ID NO:4. Joining theacridinium ester label to the probe may be carried out in accordancewith the teachings of Arnold et al. in U.S. Pat. Nos. 5,185,439 and6,031,091.

A Cryptosporidium parvum probe of the present invention preferablycomprises an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19 or SEQ ID NO:20. One group of preferred Cryptosporidium parvumprobes comprises an oligonucleotide having or substantiallycorresponding to the base sequence of SEQ ID NO:5, SEQ ID NO:9, SEQ IDNO:13 or SEQ ID NO: 17, and preferably include an acridinium ester labeljoined to the probe by means of a non-nucleotide linker positionedbetween nucleotides 18 and 19 (reading 5′ to 3′) of SEQ ID NO:5 or SEQID NO:9 and between nucleotides 9 and 10 (reading 5′ to 3′) of SEQ IDNO:13 or SEQ ID NO:17. Another group of preferred Cryptosporidium parvumprobes comprises an oligonucleotide having or substantiallycorresponding to the base sequence of SEQ ID NO:6, SEQ ID NO:10, SEQ IDNO:14 or SEQ ID NO:18, and preferably include an acridinium ester labeljoined to the probe by means of a non-nucleotide linker positionedbetween nucleotides 11 and 12 (reading 5′ to 3′) of SEQ ID NO:6 or SEQID NO:10 and between nucleotides 9 and 10 (reading 5′ to 3′) of SEQ IDNO:14 or SEQ ID NO:18. A further group of preferred Cryptosporidiumparvum probes comprises an oligonucleotide having or substantiallycorresponding to the base sequence of SEQ ID NO:7, SEQ ID NO:11, SEQ IDNO:15 or SEQ ID NO:19, and preferably include an acridinium ester labeljoined to the probe by means of a non-nucleotide linker positionedbetween nucleotides 13 and 14 (reading 5′ to 3′) of SEQ ID NO:7 or SEQID NO:11 and between nucleotides 12 and 13 (reading 5′ to 3′) of SEQ IDNO:15 or SEQ ID NO:19. A final group of preferred Cryptosporidium parvumprobes comprises an oligonucleotide having or substantiallycorresponding to the base sequence of SEQ ID NO:8, SEQ ID NO:12, SEQ IDNO:16 or SEQ ID NO:20, and preferably include an acridinium ester labeljoined to the probe by means of a non-nucleotide linker positionedbetween nucleotides 16 and 17 (reading 5′ to 3′) of SEQ ID NO:8 or SEQID NO:12 and between nucleotides 9 and 10 (reading 5′ to 3′) of SEQ IDNO:16 or SEQ ID NO:20. Joining the acridinium ester label to the probemay be carried out in accordance with the teachings of Arnold et al. inU.S. Pat. Nos. 5,185,439 and 6,031,091.

Thus, in one aspect of the present invention a Cryptosporidiumhybridization assay probe is provided which is useful for determiningwhether Cryptosporidium organisms are present in a test sample. Theprobe comprises an oligonucleotide up to 100 bases in length whichcontains an at least 10 contiguous base region which is at least 80%complementary to an at least 10 contiguous base region present in atarget nucleic acid sequence present in a target nucleic acid derivedfrom Cryptosporidium organisms. The target sequence preferably has thebase sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4,and the probe preferentially hybridizes under stringent conditions tothe target nucleic acid over nucleic acid derived fromnon-Cryptosporidium organisms present in the test sample.

Alternatively, the Cryptosporidium probe comprises an oligonucleotide upto 100 bases in length which contains an at least 10 contiguous baseregion which is at least 80% homologous to an at least 10 contiguousbase region present in the base sequence of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3 or SEQ ID NO:4. The probe preferentially hybridizes understringent conditions to the target nucleic acid over nucleic acidderived from non-Cryptosporidium organisms present in the test sample.

In another aspect of the present invention a Cryptosporidium parvumhybridization assay probe is provided which is useful for determiningwhether Cryptosporidium parvum organisms are present in a test sample.The probe comprises an oligonucleotide up to 100 bases in length whichcontains an at least 10 contiguous base region which is at least 80%complementary to an at least 10 contiguous base region present in atarget nucleic acid sequence present in a target nucleic acid derivedfrom Cryptosporidium parvum organisms. The target sequence preferablyhas the base sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19 or SEQ ID NO:20, and the probe preferentiallyhybridizes under stringent conditions to the target nucleic acid overnucleic acid derived from non-Cryptosporidium parvum organisms in thetest sample.

Alternatively, the Cryptosporidium parvum probe comprises anoligonucleotide up to 100 bases in length which contains an at least 10contiguous base region which is at least 80% homologous to an at least10 contiguous base region present in the base sequence of SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20. Theprobe preferentially hybridizes under stringent conditions to the targetnucleic acid over nucleic acid derived from non-Cryptosporidium parvumorganisms present in the test sample.

Once synthesized, the probes may be labeled with a detectable label orreporter group by any well-known method. (See, e.g., SAMBROOK ET AL.,supra, ch. 10.) The probe may be labeled with a detectable moiety suchas a radioisotope, antigen or chemiluminescent moiety to facilitatedetection of the target sequence. Useful labels include radioisotopes aswell as non-radioactive reporting groups. Isotopic labels include ³H,35S, ³²P, ¹²⁵I, ⁵⁷Co and ¹⁴C. Isotopic labels can be introduced into anoligonucleotide by techniques known in the art such as nick translation,end labeling, second strand synthesis, reverse transcription and bychemical methods. When using radiolabeled probes, hybridization can bedetected by techniques such as autoradiography, scintillation countingor gamma counting. The chosen detection method depends on the particularradioisotope used for labeling.

Non-isotopic materials can also be used for labeling and may beintroduced internally between nucleotides or at an end of theoligonucleotide. Modified nucleotides may be incorporated enzymaticallyor chemically. Chemical modifications of the oligonucleotide may beperformed during or after synthesis of the oligonucleotide usingtechniques known in the art. For example, through use of non-nucleotidelinker groups disclosed by Arnold et al. in U.S. Pat. No. 6,031,091.Non-isotopic labels include fluorescent molecules, chemiluminescentmolecules, fluorescent chemiluminescent molecules, phosphorescentmolecules, electrochemiluminescent molecules, chromophores, enzymes,enzyme cofactors, enzyme substrates, dyes and haptens or other ligands.Another useful labeling technique is a base sequence that is unable tostably hybridize to the target nucleic acid under stringent conditions.Probes of the present invention are preferably labeled with anacridinium ester. (Acridinium ester labeling is disclosed by Arnold etal. in U.S. Pat. No. 5,185,439.)

The selected hybridization assay probe can then be brought into contactwith a test sample suspected of containing Cryptosporidium orCryptosporidium parvum organisms. Generally, the test sample is from asource which also contains unknown organisms. After bringing the probeinto contact with the test sample, the test sample can be incubatedunder conditions permitting preferential hybridization of the probe to atarget nucleic acid derived from Cryptosporidium or Cryptosporidiumparvum organisms which may be present in the test sample in the presenceof nucleic acid derived from other organisms in the test sample.

The probe may also be combined with one or more unlabeled helper probesto facilitate binding to target nucleic acid derived fromCryptosporidium or Cryptosporidium parvum organisms. After the probe hashybridized to target nucleic acid present in the test sample, theresulting hybrid may be separated and detected by various techniqueswell known in the art, such as hydroxyapatite adsorption and radioactivemonitoring. Other techniques include those which involve selectivelydegrading label associated with unhybridized probe and then measuringthe amount of remaining label associated with hybridized probe, asdisclosed in U.S. Pat. No. 5,283,174. The inventors particularly preferthis latter technique.

E. Helper Probes Used in the Detection of Cryptosporidium

Another embodiment of this invention relates to novel helper probes. Asmentioned above, helper probes can be used to facilitate hybridizationof hybridization assay probes to their intended target nucleic acids, sothat the hybridization assay probes more readily form probe:targetnucleic acid duplexes than they would in the absence of helper probes.(Helper probes are disclosed by Hogan et al., “Means and Method forEnhancing Nucleic Acid Hybridization,” U.S. Pat. No. 5,030,557.) Eachhelper probe contains an oligonucleotide that is sufficientlycomplementary to a target nucleic acid sequence to form a helperprobe:target nucleic acid duplex under stringent hybridization assayconditions. The stringent hybridization assay conditions employed with agiven helper probe are determined by the conditions used forpreferentially hybridizing the associated hybridization assay probe tothe target nucleic acid.

Regions of single stranded RNA and DNA can be involved in secondary andtertiary structures even under stringent hybridization assay conditions.Such structures can sterically inhibit or block hybridization of ahybridization assay probe to a target nucleic acid. Hybridization of thehelper probe to the target nucleic acid alters the secondary andtertiary structure of the target nucleic acid, thereby rendering thetarget region more accessible by the hybridization assay probe. As aresult helper probes enhance the kinetics and/or the melting temperatureof the hybridization assay probe:target nucleic acid duplex. Helperprobes are generally selected to hybridize to nucleic acid sequenceslocated near the target region of the hybridization assay probe.

Helper probes which can be used with the Cryptosporidium hybridizationassay probes of the present invention are targeted to nucleic acidsequences within Cryptosporidium-derived nucleic acid. Likewise, helperprobes which can be used with the Cryptosporidium parvum hybridizationassay probes of the present invention are targeted to nucleic acidsequences within Cryptosporidium parvum-derived nucleic acid. Eachhelper probe comprises an oligonucleotide which targets a base regionpresent in a target nucleic acid. Preferably, the helper probe containsan at least 10 contiguous base region which his at least 80%complementary to an at least 10 contiguous base region present in atarget nucleic acid sequence present in target nucleic acid derived fromCryptosporidium or Cryptosporidium parvum organisms, as appropriate.Helper probes and their associated hybridization assay probes havedifferent target nucleic acid sequences contained within the same targetnucleic acid. The helper probes of the present invention are preferablyoligonucleotides up to 100 bases in length, more preferably from 12 to50 bases in length, and even more preferably from 18 to 35 bases inlength. Alternatively, the helper probes may be at least 90%complementary, or even perfectly complementary, to their target regions.

Examples of Cryptosporidium helper probes which may be useful in thepresent invention are those comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27 or SEQ ID NO:28.

Examples of Cryptosporidium parvum helper probes which may be useful inthe present invention are those comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44. Whilethe inventors found that helper probes consisting of the nucleotide basesequences of SEQ ID NO:38 (20 pmol) and SEQ ID NO:39 (20 pmol), whenprovided to a 200 μl sample solution containing 100 pmol probe andtarget nucleic acid ranging in amounts from 0.1 fmol to 100 fmol, didnot appear to facilitate hybridization of the probe to the target, it isstill believed that optimization of the concentration of these helperprobes may result in improved hybridization of a hybridization assayprobe consisting of the nucleotide base sequence of SEQ ID NO:13 or SEQID NO: 17 to a complementary sequence contained in a Cryptosporidiumparvum 18S rRNA target nucleic acid.

When the helper probes are used in combination with Cryptosporidiumhybridization assay probes, the preferred hybridization assay probecomprises an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ IDNO:4, and the preferred helper probe comprises an oligonucleotide havingor substantially corresponding to the base sequence of SEQ ID NO:21, SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27 or SEQ ID NO:28.

In one preferred embodiment of this combination, the Cryptosporidiumhybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 or SEQ ID NO:4, and the helper probe comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27.

In a further preferred embodiment of this combination, theCryptosporidium hybridization assay probe comprises an oligonucleotidehaving or substantially corresponding to the base sequence of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and the helper probecomprises an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 or SEQ IDNO:28.

Another preferred combination includes at least two helper probes whenthe Cryptosporidium hybridization assay probe comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. Inthis combination, the first helper probe preferably comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 or SEQ ID NO:27,and the second helper probe preferably comprises an oligonucleotidehaving or substantially corresponding to the base sequence of SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28.

When helper probes are used in combination with Cryptosporidium parvumhybridization assay probes, the following combinations are preferred.When the hybridization assay probe comprises an oligonucleotide havingor substantially corresponding to the base sequence of SEQ ID NO:5, SEQID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:17 or SEQ ID NO:18, the helper probe preferably comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43 or SEQ ID NO:44.

In one embodiment of this combination, the Cryptosporidium parvumhybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:17 or SEQ ID NO:18, and the helper probe comprises an oligonucleotidehaving or substantially corresponding to the base sequence of SEQ IDNO:29, SEQ ID NO:33, SEQ ID NO:37 or SEQ ID NO:41.

In a further embodiment of this combination, the Cryptosporidium parvumhybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:17 or SEQ ID NO:18, and the helper probe comprises an oligonucleotidehaving or substantially corresponding to the base sequence of SEQ IDNO:30, SEQ ID NO:34, SEQ ID NO:38 or SEQ ID NO:42.

In another embodiment of this combination, the Cryptosporidium parvumhybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:17 or SEQ ID NO:18, and the helper probe comprises an oligonucleotidehaving or substantially corresponding to the base sequence of SEQ IDNO:31, SEQ ID NO:35, SEQ ID NO:39 or SEQ ID NO:43.

In yet a further embodiment of this combination, the Cryptosporidiumparvum hybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:17 or SEQ ID NO:18, and the helper probe comprises an oligonucleotidehaving or substantially corresponding to the base sequence of SEQ IDNO:32, SEQ ID NO:36, SEQ ID NO:40 or SEQ ID NO:44.

Another preferred combination includes at least two helper probes whenthe Cryptosporidium parvum hybridization assay probe comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:17 or SEQ ID NO:18. In this combination,the first helper probe preferably comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:41 or SEQ ID NO:42, and the second helper probe preferably comprisesan oligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:36, SEQID NO:39, SEQ ID NO:40, SEQ ID NO:43 or SEQ ID NO:44.

In one embodiment of this combination, the Cryptosporidium parvumhybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:17 or SEQ ID NO:18. The first helper probe comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37 or SEQ ID NO:41,and the second helper probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:31, SEQ IDNO:35, SEQ ID NO:39 or SEQ ID NO:43.

In a further embodiment of this combination, the Cryptosporidium parvumhybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:17 or SEQ ID NO:18. The first helper probe comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37 or SEQ ID NO:41,and the second helper probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:32, SEQ IDNO:36, SEQ ID NO:40 or SEQ ID NO:44.

In another embodiment of this combination, the Cryptosporidium parvumhybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:17 or SEQ ID NO:18. The first helper probe comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38 or SEQ ID NO:42,and the second helper probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:31, SEQ IDNO:35, SEQ ID NO:39 or SEQ ID NO:43.

In yet a further embodiment of this combination, the Cryptosporidiumparvum hybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:17 or SEQ ID NO:18. The first helper probe comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38 or SEQ ID NO:42,and the second helper probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:32, SEQ IDNO:36, SEQ ID NO:40 or SEQ ID NO:44.

In yet another preferred combination, the Cryptosporidium parvumhybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:7, SEQ IDNO:11, SEQ ID NO:15 or SEQ ID NO:19, and the helper probe comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQID NO:37, SEQ ID NO:38, SEQ ID NO:41 or SEQ ID NO:42.

In one embodiment of this combination, the Cryptosporidium parvumhybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:7, SEQ IDNO:11, SEQ ID NO:15 or SEQ ID NO:19, and the helper probe comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:29, SEQ ID NO:33, SEQ ID NO:37 or SEQ ID NO:41.

In further embodiment of this combination, the Cryptosporidium parvumhybridization assay probe comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:7, SEQ IDNO:11, SEQ ID NO:15 or SEQ ID NO:19, and the helper probe comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38 or SEQ ID NO:42.

F. Nucleic Acid Compositions

In another related aspect, the present invention features compositionscomprising a nucleic acid hybrid formed between a hybridization assayprobe and a target nucleic acid (“probe:target”) under stringenthybridization assay conditions. One use of the hybrid formed between aprobe and a target nucleic acid is to provide an indication of thepresence or amount of a target organism or group of organisms in a testsample. For example, acridinium ester (AE) present in nucleic acidhybrids is resistant to hydrolysis in an alkali solution, whereas AEpresent in single-stranded nucleic acid is susceptible to hydrolysis inan alkali solution (see U.S. Pat. No. 5,238,174). Thus, the presence oftarget nucleic acids can be detected, after the hydrolysis of theunbound AE-labeled probe, by measuring chemiluminescence of acridiniumester remaining associated with the nucleic acid hybrid.

The present invention also contemplates compositions comprising nucleicacid hybrids formed between a helper probe and a target nucleic acid(“helper probe:target”) under stringent hybridization assay conditions.One use of the hybrid formed between a helper probe and a target nucleicacid is to make available a particular nucleic acid sequence forhybridization. For example, a hybrid formed between a helper probe and atarget nucleic acid may render a nucleic acid sequence available forhybridization with a hybridization assay probe. A full description ofthe use of helper probes is provided by Hogan et al. in U.S. Pat. No.5,030,557.

The present invention also features compositions comprising a nucleicacid formed between an amplification primer and a target nucleic acid(“primer:target”) under amplification conditions. One use of the hybridformed between a primer and a target nucleic acid is to provide aninitiation site for a nucleic acid polymerase at the 3′ end of theamplification primer. For example, a hybrid may form an initiation sitefor reverse transcriptase, DNA polymerases such as Taq polymerase or T4DNA polymerase, and RNA polymerases such as T7 polymerase, SP6polymerase, T3 polymerase and the like.

Compositions of the present invention include compositions fordetermining the presence or amount of Cryptosporidium or Cryptosporidiumparvum organisms in a test sample comprising a nucleic acid hybridformed between a target nucleic acid derived from a Cryptosporidiumorganism and an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ ID NO:68.

The present invention also contemplates compositions for determining thepresence or amount of Cryptosporidium organisms in a test samplecomprising a nucleic acid hybrid formed between a target nucleic acidderived from a Cryptosporidium organism and a hybridization assay probecomprising an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ IDNO:4. In another embodiment, these probe:target compositions may furthercomprise a helper probe hybridized to the Cryptosporidium-derived targetnucleic acid, where the helper probe comprises an oligonucleotide havingor substantially corresponding to the base sequence of SEQ ID NO:21, SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27 or SEQ ID NO:28. Preferred hybridization assay probe and helperprobe combinations for forming this latter group of nucleic acid hybridcompositions are those of the probe mix combinations described aboveunder the heading “Helper Probes Used in the Detection ofCryptosporidium.”

The present invention further contemplates compositions for determiningthe presence or amount of Cryptosporidium parvum organisms in a testsample comprising a nucleic acid hybrid formed between a target nucleicacid derived from a Cryptosporidium parvum organism and a hybridizationassay probe comprising an oligonucleotide having or substantiallycorresponding to the base sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20. In anotherembodiment, these probe:target compositions may further comprise ahelper probe hybridized to the Cryptosporidium parvum-derived targetnucleic acid, where the helper probe comprises an oligonucleotide havingor substantially corresponding to the base sequence of SEQ ID NO:29, SEQID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 or SEQ ID NO:44.Preferred hybridization assay probe and helper probe combinations forforming this latter group of nucleic acid hybrid compositions are thoseof the probe mix combinations described above under the heading “HelperProbes Used in the Detection of Cryptosporidium.”

The present invention also contemplates compositions for amplifying atarget sequence present in a target nucleic acid derived from aCryptosporidium organism, where the compositions comprise a nucleic acidhybrid formed between the target nucleic acid and an amplificationprimer comprising an oligonucleotide having or substantiallycorresponding to the base sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:67 or SEQ ID NO:68. Preferred amplification primer combinations forforming these nucleic acid hybrid compositions are those described aboveunder the heading “Amplification of Cryptosporidium Ribosomal NucleicAcid.”

G. Assay Methods

The present invention contemplates various methods for assaying for thepresence or amount of nucleic acid derived from Cryptosporidium orCryptosporidium parvum organisms in a test sample. One skilled in theart will understand that the exact assay conditions, probes and/orprimers used will vary depending on the particular assay format used andthe source of the sample.

One aspect of the present invention relates to a method for determiningthe presence or amount of Cryptosporidium organisms in a test sample bycontacting the test sample under stringent hybridization assayconditions with a hybridization assay probe capable of preferentiallyhybridizing under stringent hybridization assay conditions to aCryptosporidium-derived target nucleic acid over nucleic acids fromnon-Cryptosporidium organisms present in the test sample. In thismethod, the target nucleic acid contains a base sequence having orsubstantially corresponding to the base sequence of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3 or SEQ ID NO:4. (Depending on the source, the testsample may contain unknown organisms that the probes of this method candistinguish over.) Preferred probes for use in this method comprise anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

In a preferred embodiment, the method for determining the presence oramount of Cryptosporidium organisms in a test sample may also includethe step of contacting the test sample with one or more helper probesfor facilitating hybridization of the probe to the target nucleic acid.While the helper probes may be added to the sample before or after theaddition of the hybridization assay probe, the helper probes andhybridization assay probe are preferably provided to the test sample atthe same time. Preferred helper probes for use in this method comprisean oligonucleotide having or substantially corresponding to the basesequence SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28. Particularcombinations of hybridization assay probes and helper probes which canbe used in this method are set forth above under the heading “HelperProbes Used in the Detection of Cryptosporidium.”

Another aspect of the present invention relates to a method fordetermining the presence or amount of Cryptosporidium parvum organismsin a test sample by contacting the test sample under stringenthybridization assay conditions with a hybridization assay probe capableof preferentially hybridizing under stringent hybridization assayconditions to a Cryptosporidium parvum-derived target nucleic acid overnucleic acids from non-Cryptosporidium parvum organisms present in thetest sample. In this method, the target nucleic acid contains a basesequence having or substantially corresponding to the base sequence ofSEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ IDNO:20. (Depending on the source, the test sample may contain unknownorganisms that the probes of this method can distinguish over.)Preferred probes for use in this method comprise an oligonucleotidehaving or substantially corresponding to the base sequence of SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

In a preferred embodiment, the method for determining the presence oramount of Cryptosporidium parvum organisms in a test sample may alsoinclude the step of contacting the test sample with one or more helperprobes for facilitating hybridization of the probe to the target nucleicacid. While the helper probes may be added to the sample before or afterthe addition of the hybridization assay probe, the helper probes andhybridization assay probe are preferably provided to the test sample atthe same time. Preferred helper probes for use in this method comprisean oligonucleotide having or substantially corresponding to the basesequence SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43 or SEQ ID NO:44. Particular combinations of hybridization assayprobes and helper probes which can be used in this method are set forthabove under the heading “Helper Probes Used in the Detection ofCryptosporidium.”

Yet another aspect of the present invention relates to a method foramplifying Cryptosporidium-derived nucleic acid in a test sample bycontacting the test sample under amplification conditions with one ormore amplification primers which, when contacted with a nucleic acidpolymerase, will bind to or cause elongation through a nucleic acidregion having the base sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:67 or SEQ ID NO:68. Preferred amplification primers for use in thismethod comprise an oligonucleotide having or substantially correspondingto the base sequence of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ IDNO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ IDNO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ IDNO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67 or SEQ IDNO:68, where the amplification primer optionally includes a nucleic acidsequence recognized by an RNA polymerase or which enhances initiation orelongation by an RNA polymerase. Particular combinations ofamplification primers which can be used in this method are set forthabove under the heading “Amplification of Cryptosporidium RibosomalNucleic Acid.”

In a preferred embodiment, the method for amplifyingCryptosporidium-derived nucleic acid in a test sample further includesthe step of contacting the test sample under stringent hybridizationassay conditions with a hybridization assay probe capable ofpreferentially hybridizing under stringent hybridization assayconditions to an amplified Cryptosporidium target nucleic acid overnucleic acids from non-Cryptosporidium organisms present in the testsample. While the test sample is generally contacted with thehybridization assay probe after a sufficient period for amplificationhas passed, the amplification primers and hybridization assay probe maybe added to the sample in any order, especially where the hybridizationassay probe is a self-hybridizing probe, such as a Molecular Torch or aMolecular Beacon discussed supra. This step of contacting the testsample with a hybridization assay probe is performed so that thepresence or amount of Cryptosporidium organisms in the test sample canbe determined. Preferred probes for use in this method comprise anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. Theprobes may further include a label to facilitate detection in the testsample.

In one preferred embodiment, this method is carried out with a set of atleast two amplification primers for amplifying Cryptosporidium-derivednucleic acid which includes a first amplification primer comprising anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57 or SEQ ID NO:63,and a second amplification primer comprising an oligonucleotide havingor substantially corresponding to the base sequence of SEQ ID NO:48, SEQID NO:54, SEQ ID NO:60 or SEQ ID NO:66. The hybridization assay probeused to specifically detect amplified Cryptosporidium nucleic acid inthe test sample comprises an oligonucleotide having or substantiallycorresponding to the base sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3 or SEQ ID NO:4.

In another preferred embodiment, this method is carried out with a setof at least two amplification primers for amplifyingCryptosporidium-derived nucleic acid which includes a firstamplification primer comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:46, SEQ IDNO:52, SEQ ID NO:58 or SEQ ID NO:64, and a second amplification primercomprising an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:60 or SEQ IDNO:66. The hybridization assay probe used to specifically detectamplified Cryptosporidium nucleic acid in the test sample comprises anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

In a preferred embodiment, the method for amplifyingCryptosporidium-derived nucleic acid in a test sample further includesthe step of contacting the test sample under stringent hybridizationassay conditions with a hybridization assay probe capable ofpreferentially hybridizing under stringent hybridization assayconditions to an amplified Cryptosporidium parvum target nucleic acidover nucleic acids from non-Cryptosporidium parvum organisms present inthe test sample. While the test sample is generally contacted with thehybridization assay probe after a sufficient period for amplificationhas passed, the amplification primers and hybridization assay probe maybe added to the sample in any order, especially where the hybridizationassay probe is a self-hybridizing probe, such as a Molecular Torch or aMolecular Beacon discussed supra. This step of contacting the testsample with a hybridization assay probe is performed so that thepresence or amount of Cryptosporidium parvum organisms in the testsample can be determined. Preferred probes for use in this methodcomprise an oligonucleotide having or substantially corresponding to thebase sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19 or SEQ ID NO:20. The probes may further include a label tofacilitate detection in the test sample.

In one preferred embodiment, this method is carried out with a set of atleast two amplification primers for amplifying Cryptosporidium-derivednucleic acid which includes a first amplification primer comprising anoligonucleotide having or substantially corresponding to the basesequence of SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:57 or SEQ ID NO:63,and a second amplification primer comprising an oligonucleotide havingor substantially corresponding to the base sequence of SEQ ID NO:47, SEQID NO:53, SEQ ID NO:59 or SEQ ID NO:65. The hybridization assay probeused to specifically detect amplified Cryptosporidium parvum nucleicacid in the test sample comprises an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18 or SEQ IDNO:19.

In another preferred embodiment, this method is carried out with a setof at least two amplification primers for amplifyingCryptosporidium-derived nucleic acid which includes a firstamplification primer comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:45, SEQ IDNO:51, SEQ ID NO:57 or SEQ ID NO:63, and a second amplification primercomprising an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:60 or SEQ IDNO:66. The hybridization assay probe used to specifically detectamplified Cryptosporidium parvum nucleic acid in the test samplecomprises an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

In yet another preferred embodiment, this method is carried out with aset of at least two amplification primers for amplifyingCryptosporidium-derived nucleic acid which includes a firstamplification primer comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:46, SEQ IDNO:52, SEQ ID NO:58 or SEQ ID NO:64, and a second amplification primercomprising an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:47, SEQ ID NO:53, SEQ ID NO:59 or SEQ IDNO:65. The hybridization assay probe used to specifically detectamplified Cryptosporidium parvum nucleic acid in the test samplecomprises an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

In still another preferred embodiment, this method is carried out with aset of at least two amplification primers for amplifyingCryptosporidium-derived nucleic acid which includes a firstamplification primer comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:46, SEQ IDNO:52, SEQ ID NO:58 or SEQ ID NO:64, and a second amplification primercomprising an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:60 or SEQ IDNO:66. The hybridization assay probe used to specifically detectamplified Cryptosporidium parvum nucleic acid in the test samplecomprises an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:1O, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

In a further preferred embodiment, this method is carried out with a setof at least two amplification primers for amplifyingCryptosporidium-derived nucleic acid which includes a firstamplification primer comprising an oligonucleotide having orsubstantially corresponding to the base sequence of SEQ ID NO:49, SEQ IDNO:55, SEQ ID NO:61 or SEQ ID NO:67, and a second amplification primercomprising an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:50, SEQ ID NO:56, SEQ ID NO:62 or SEQ IDNO:68. The hybridization assay probe used to specifically detectamplified Cryptosporidium parvum nucleic acid in the test samplecomprises an oligonucleotide having or substantially corresponding tothe base sequence of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16 or SEQ IDNO:20.

H. Diagnostic Systems

The present invention also contemplates diagnostic systems in kit form.A diagnostic system of the present invention may include a kit whichcontains, in an amount sufficient for at least one assay, any of thehybridization assay probes, helper probes and/or amplification primersof the present invention in a packaging material. Typically, the kitswill also include instructions recorded in a tangible form (e.g.,contained on paper or an electronic medium) for using the packagedprobes and/or primers in an amplification and/or detection assay fordetermining the presence or amount of Cryptosporidium or Cryptosporidiumparvum organisms in a test sample.

The various components of the diagnostic systems may be provided in avariety of forms. For example, the required enzymes, the nucleotidetriphosphates, the probes and/or primers may be provided as alyophilized reagent. These lyophilized reagents may be pre-mixed beforelyophilization so that when reconstituted they form a complete mixturewith the proper ratio of each of the components ready for use in theassay. In addition, the diagnostic systems of the present invention maycontain a reconstitution reagent for reconstituting the lyophilizedreagents of the kit. In preferred kits for amplifying target nucleicacid derived from Cryptosporidium organisms, the enzymes, nucleotidetriphosphates and required cofactors for the enzymes are provided as asingle lyophilized reagent that, when reconstituted, forms a properreagent for use in the present amplification methods. In these kits, alyophilized primer reagent may also be provided. In other preferredkits, lyophilized probe reagents are provided.

Typical packaging materials would include solid matrices such as glass,plastic, paper, foil, micro-particles and the like, capable of holdingwithin fixed limits hybridization assay probes, helper probes and/oramplification primers of the present invention. Thus, for example, thepackaging materials can include glass vials used to containsub-milligram (e.g., picogram or nanogram) quantities of a contemplatedprobe or primer, or they can be microtiter plate wells to which probesor primers of the present invention have been operatively affixed, i.e.,linked so as to be capable of participating in an amplification and/ordetection method of the present invention.

The instructions will typically indicate the reagents and/orconcentrations of reagents and at least one assay method parameter whichmight be, for example, the relative amounts of reagents to use peramount of sample. In addition, such specifics as maintenance, timeperiods, temperature and buffer conditions may also be included.

The diagnostic systems of the present invention contemplate kits havingany of the hybridization assay probes, helper probes and/oramplification primers described herein, whether provided individually orin one of the preferred combinations described above, for use inamplifying and/or determining the presence or amount of Cryptosporidiumor Cryptosporidium parvum organisms in a test sample.

I. EXAMPLES

Examples are provided below illustrating different aspects andembodiments of the invention. Skilled artisans will appreciate thatthese examples are not intended to limit the invention to the specificembodiments described therein.

A. Isolation and Purification of Cryptosporidium Nucleic Acid

To obtain Cryptosporidium ribosomal RNA, Cryptosporidium oocysts arefirst isolated from a sample by centrifuging the sample in an Eppendorf1.5 ml micro test tube (Brinkmann Instruments, Inc., Westbury, N.Y.;Cat. No. 22 60 002 8) at 7,000 rpm for 5 minutes using an Eppendorfmicrocentrifuge (Brinkmann Instruments, Inc.; Cat. No. 22 62 120-3). Thesupernatant is then aspirated off and the remaining pellet isresuspended in 200 μl lysis/binding buffer provided with Ambion'sRNAaqueous™ kit (Ambion, Inc, Austin, Tex.; Cat. No. AM-1912).

Approximately 1.0 mm zirconia/silica beads (BioSpec Products, Inc.,Bartlesville, Okla; Cat. No. 11079110Z) are added to a 2.0 mlmicrocentrifuge vial (BioSpec Products, Inc.; Cat. No. 10832), fillingthe vial about three quarters full (1.5 ml). Then, 500 μl ofdiethylpyrocarbonate (DEPC)-treated water (Ambion, Inc.; Cat. No. 9922)is added to the vial and the vial is sealed and inverted by hand severaltimes prior to aspirating the DEPC-treated water from the vial.

The resuspended pellet is removed from the micro test tube and added tothe bead-containing vial. Additional lysis/binding buffer is added tocompletely fill the vial before the vial is again sealed and positionedin a BeadBeater™ (BioSpec Products, Inc.; Cat. No. 3110BX ), which isrun for 2 minutes at 5,000 rpm. The vial is removed from the BeadBeater™and placed on ice for 4 minutes before being subjected to a second runin the BeadBeater™ for 2 minutes at 5,000 rpm. The vial is again placedon ice for 4 minutes.

The supernatant is pipetted from the vial, thereby separating it fromthe beads, and added to a new 2 ml microcentrifuge vial (BioSpecProducts, Inc.; Cat. No. 10832). Also added to this vial is ethanol(64%) provided with the RNAqueous™ kit in equal volume with thesupernatant before the vial is sealed and inverted by hand several timesto ensure thorough mixing of the supernatant and ethanol. This mixtureis then added in 600 μl aliquots to the filter cartridges of collectiontubes provided with the RNAqueous™ kit. The collection tubes are thencentrifuged for 30 seconds at 13,000 rpm in the Eppendorfmicrocentrifuge, the eluent is aspirated off, and 700 μl of WashSolution I from the RNAqueous ™ kit is added to each filter cartridge.The collection tubes are once more centrifuged at 13,000 rpm for 30seconds, the eluent is removed, and 500 μl of Wash Solution 2/3 from theRNAqueous™ kit is added to the filter cartridges. This lastcentrifugation and treatment with Wash Solution 2/3 is repeated toensure removal of the ethanol. The collection tubes are then centrifugedat 13,000 rpm for 30 seconds, and the eluent is removed prior tocentrifuging the collection tubes at 13,000 rpm for 2 minutes.

The filter cartridges are then placed in another set of collection tubesprovided with the RNAqueous™ kit, to which 50 μl of an elution solutionprovided with the RNAqueous™ kit (pre-heated to 95° C.) is added tothese new collection tubes. The collection tubes are centrifuged for 1minute at 13,000 rpm in the microcentrifuge before another 50 μl of thepre-heated elution solution is added to the collection tubes. A final 1minute centrifugation at 13,000 rpm is performed, and the eluent fromthese last two centrifugations contains the purified RNA.

B. Hybridization Assay Probes

Hybridization assay probes specific for nucleic acid fromCryptosporidium or Cryptosporidium parvum were identified from thepublished sequences indicated supra and a determined 18S rRNA sequencefor Cryptosporidium parvum. Probes specific for Cryptosporidium wereidentified by comparing 18S rRNA sequences of Cryptosporidium parvum,Cryptosporidium muris, Cryptosporidium baileyi and Cryptosporidiumwrairi with 18S rRNA sequences of Escherichia coli, Cyclosporacayetanensis, Sarcocystis hominis, Entamoeba histolytica and Eimeriapraecox. And to identify probes specific for Cryptosporidium parvum, an18S rRNA sequence of Cryptosporidium parvum was compared with 18S rRNAsequences of Cryptosporidium muris, Cryptosporidium bailyei andCryptosporidium wrairi. Regions of variability were identified whichcould be tested to verify specificity.

Hybridization assay probes having the base sequences of SEQ ID NO. 1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:18 are featured in theexamples described below. The probes were synthesized with anon-nucleotide linker, as disclosed by Arnold et al. in U.S. Pat. No.6,031,091, and labeled with a chemiluminescent acridinium ester (AE), asdisclosed by Arnold et al. in U.S. Pat. No. 5,185,439. The reactivityand specificity of the probes for Cryptosporidium and Cryptosporidiumparvum nucleic acid are demonstrated using the Homogenous ProtectionAssay (HPA) disclosed by Arnold et al. in U.S. Pat. No. 5,283,174.Results are given in relative light units (RLU), which is a measure ofthe photons detected by a luminometer.

C. Reagents

Various reagents are identified in the examples below, which include ahybridization reagent, a selection reagent, an amplification reagent, areconstitution buffer, an enzyme reagent, an enzyme dilution buffer andan oil reagent. The formulations and pH values (where relevant) of thesereagents are as follows.

Hybridization Reagent: The “Hybridization Reagent” of the followingexamples is made up of 50 mM succinic acid, 1% (w/v) lithium laurylsulfate (LLS), 7.5 mM aldrithiol-2, 0.6 M LiCl, 115 mM LiOH, 10 mMethylenediaminetetraacetic acid (EDTA), 10 mM ethylene glycol N, N, N′,N′-tetraacetic acid (EGTA) and 1.5% (v/v) ethyl alcohol (absolute), pHto 4.7.

Selection Reagent: The “Selection Reagent” of the following examples ismade up of 600 mM boric acid, 240 mM NaOH and 1% (v/v) TRITON® X-100, pHto 8.5.

Amplification Reagent: The “Amplification Reagent” of the followingexamples is made up of 4 mM each of ATP, GTP, UTP and CTP, 1 mM each ofdATP, dGTP, dTTP and dCTP, 40 mM trizma base, pH 7.5, 25 mM MgCl₂, 17.5mM KCl and 5% (w/v) polyvinylpyrrolidone. The Amplification Reagent isreconstituted with 1.5 ml distilled, deionized water.

Enzyme Reagent: The “Enzyme Reagent” of the following examples is madeup of 125 mM N-acetyl-L-cysteine (NALC), 0.2% (v/v) TRITON® X-102, 20 mMN-2-hydroxyethelpiperazine-N′-2-ethanesulfonic acid (HEPES), pH 7.5, 0.1mM EDTA, 0.1 mM zinc acetate, 0.2 M trehalose, 2000 units Moloney MurineLeukemia Virus (“MMLV”) reverse transcriptase and 2000 units T7 RNApolymerase. (One “unit” of activity is defined as the synthesis andrelease of 5.75 fmol cDNA in 15 minutes at 37° C. for MMLV reversetranscriptase, and for T7 RNA polymerase, one “unit” of activity isdefined as the production of 5.0 fmol RNA transcript in 20 minutes at37° C.) The Enzyme Reagent is reconstituted with 1.5 mL Enzyme DiluentBuffer.

Enzyme Dilution Buffer: The “Enzyme Dilution Buffer” of the followingexamples is made up of 140 mM trizma base, pH 8.0, 1 mM EDTA, 10% (v/v)TRITON® X-102, 70 mM KCl and 20% (v/v) glycerol.

Detection Reagents: The “Detection Reagents” of the following examplescomprise Detect Reagent I, which contains 0.1% (v/v) hydrogen peroxideand 1 mM nitric acid, and Detect Reagent II, which contains 1N sodiumhydroxide and a surfactant component.

Oil Reagent: The “Oil Reagent” of the following examples is a mineraloil.

Example 1 Specific Detection of Cryptosporidium Nucleic Acid

This example illustrates the ability of a probe mixture containing anacridinium ester-labeled hybridization assay probe targeted toCryptosporidium rRNA to selectively detect Cryptosporidium species inthe presence of non-Cryptosporidium organisms. A hybridization assayprobe having the base sequence of SEQ ID NO:3 is synthesized, asdescribed above, to include a non-nucleotide linker positioned betweennucleotides 11 and 12 (reading 5′ to 3′). Additionally, unlabeled helperprobes having the base sequences of SEQ ID NO:27 and SEQ ID NO:28 areincluded in the probe mixture to facilitate hybridization of thehybridization assay probe to Cryptosporidium nucleic acid. Except forthe first nucleotide (reading 5′ to 3′), which is substituted with thecorresponding deoxynucleotide, each nucleotide of the helper probes is aribonucleotide modified to include a 2′-O-methyl substitution to theribofuranosyl moiety.

The specificity of the hybridization assay probe for Cryptosporidiumnucleic acid is determined using a panel of duplicate sample sets, eachset containing 100 ng of purified rRNA (50 ng per sample) from anisolate chosen from the group consisting of Escherichia coli, Cyclosporacayetanensis, Sarcocystis hominis, Entamoeba histolytica, Eimeriapraecox, Cryptosporidium parvum and at least one of Cryptosporidiummuris, Cryptosporidium baileyi and Cryptosporidium wrairi.Cryptosporidium parvum and Cryptosporidium muris isolates can beobtained from Waterborne Incorporated of New Orleans, La. (Part No. P102for Cryptosporidium parvum and Part No. P104 for Cryptosporidium muris).A set of negative control samples is included to determine backgroundlevels.

Each sample is provided to a 12×75 mm polypropylene tube (Gen-ProbeIncorporated; Cat. No. 2440). Additionally, each tube is provided with0.1 pmol of the hybridization assay probe, 5.0 pmol of each helperprobe, and 100 μl 1× Hybridization Reagent. To permit hybridization, thetubes are incubated at 60° C. in a circulating water bath (PrecisionScientific, Winchester, Va.; Model 260; Cat. No. 51221035) for 30minutes. Following hybridization, 300 μl Selection Reagent is added toeach tube and the tubes are incubated at 60° C. in the circulating waterbath for 10 minutes to hydrolyze acridinium ester labels associated withunhybridized probe. Samples are cooled on ice for 1 minute prior tobeing analyzed in a LEADER® 450i luminometer equipped with automaticinjection of the Detection Regents. A net RLU value greater than 10,000RLU is considered to be a positive result, and a net RLU value less than10,000 RLU is considered to be a negative result. Net RLU values arebased on the average RLU value of each sample set minus the average RLUvalue for the negative control set (i.e., background signal).

Example 2 Amplification and Detection of Cryptosporidium Nucleic Acid

This example illustrates the amplification of a target sequence ofCryptosporidium rRNA and detection of the amplified rRNA using ahybridization assay probe specific for Cryptosporidium-derived nucleicacid. In particular, a Cryptosporidium hybridization assay probe havingthe base sequence of SEQ ID NO:1 is synthesized, as described above, toinclude a non-nucleotide linker positioned between nucleotides 11 and 12(reading 5′ to 3′). This hybridization assay probe is of the same senseas the Cryptosporidium target rRNA and is used to detect product of atranscription-mediated amplification. Procedures for performing atranscription-mediated amplification are described supra and by Kacianet al. in U.S. Pat. Nos. 5,399,491 and 5,480,784. In addition, thishybridization assay probe is the opposite sense of the hybridizationassay probe of Example 1, which is believed to be specific for nucleicacid from Cryptosporidium organisms. Accordingly, the hybridizationassay probe of this example is expected to be specific for nucleic acidderived from Cryptosporidium organisms.

Ribosomal RNA from Cryptosporidium parvum and Cryptosporidium muris isseparately amplified using one of the following promoter-primer/primercombinations: (i) a promoter-primer having a 5′ end promoter basesequence of SEQ ID NO:69 and a 3′ end sense template-specific basesequence of SEQ ID NO:60, and a primer having an antisensetemplate-specific base sequence of SEQ ID NO:45; and (ii) apromoter-primer having a 5′ end promoter base sequence of SEQ ID NO:69and a 3′ end sense template-specific base sequence of SEQ ID NO:60, anda primer having an antisense template-specific base sequence of SEQ IDNO:46. Cryptosporidium parvum and Cryptosporidium muris isolates can beobtained from Waterborne Incorporated of New Orleans, La. (Part No. P102for Cryptosporidium parvum and Part No. P104 for Cryptosporidium muris).

Amplification is carried out in 12×75 mm polypropylene tubes (Gen-ProbeIncorporated; Cat. No. 2440), each containing 0 amol, 0.2 amol or 2.0amol of Cryptosporidium parvum or Cryptosporidium muris rRNA induplicate sets, 15 pmol promoter-primer, 15 pmol primer, 25 μlAmplification Reagent, and distilled, deionized water to bring the totalvolume in each tube to 75 μl. Each sample receives 200 μl Oil Reagentand is incubated at 95° C. in a dry heat bath (Gen-Probe Incorporated;Cat. No. 4006) for 10 minutes. The samples are then transferred to acirculating water bath (Lauda Dr. R. Wobser GmbH & Co. KG,Lauda-Koenigshofen, Germany; Model No. M20-S) and incubated for 5minutes at 42° C. before adding 25 μl of reconstituted Enzyme Reagent toeach tube. Following a 60 minute incubation at 42° C. in the circulatingwater bath, 100 μl 1× Hybridization Reagent containing 100 fmol of thehybridization assay probe is added to each tube, the samples areincubated for 30 minutes at 60° C. in the circulating water bath, andsignal from annealed hybridization assay probe is detected in the mannerdescribed in Example 1. Sample sets with an average RLU value greaterthan 10-fold the average RLU value for the negative control (0 amoltarget rRNA) indicate amplification of the target rRNA, and sample setswith an average RLU value less than 10-fold the average RLU for thenegative control indicate no amplification of the target rRNA.

Example 3 Specific Detection of Cryptosporidium parvum Nucleic Acid

This example illustrates the ability of a probe mixture containing anacridinium ester-labeled hybridization assay probe targeted toCryptosporidium parvum rRNA to selectively detect Cryptosporidium parvumorganisms in the presence of non-Cryptosporidium parvum organisms. Ahybridization assay probe having one of the following base sequences issynthesized, as described above: (i) SEQ ID NO:13 (non-nucleotide linkerpositioned between nucleotides 9 and 10, reading 5′ to 3′); (ii) SEQ IDNO: 15 (non-nucleotide linker positioned between nucleotides 12 and 13,reading 5′ to 3′); (iii) SEQ ID NO:16 (non-nucleotide linker positionedbetween nucleotides 9 and 10, reading 5′ to 3′); and (iv) SEQ ID NO:18(non-nucleotide linker positioned between nucleotides 9 and 10, reading5′ to 3′). Except for the first nucleotide (reading 5′ to 3′), which issubstituted with the corresponding deoxynucleotide, each nucleotide ofthe hybridization assay probe having the base sequence of SEQ ID NO:18is a ribonucleotide modified to include 2′-O-methyl substitution to theribofuranosyl moiety.

Additionally, unlabeled helper probes having the following basesequences are included in the probe mixture to facilitate hybridizationof the hybridization assay probe to Cryptosporidium parvum nucleic acid:(i) SEQ ID NO:41 or SEQ ID NO:42 for use with the hybridization assayprobe having the base sequence of SEQ ID NO:15; and (ii) any one or acombination of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44for use with the hybridization assay probe having the base sequence ofSEQ ID NO:13 or SEQ ID NO:18 (modified as indicated). Except for thefirst nucleotide (reading 5′ to 3′), which is substituted with thecorresponding deoxynucleotide, each nucleotide of the helper probes is aribonucleotide modified to include a 2′-O-methyl substitution to theribofuranosyl moiety.

The specificity of the hybridization assay probe for Cryptosporidiumparvum nucleic acid is determined using a panel of duplicate samplesets, each set containing 100 ng of purified rRNA (50 ng per sample)from an isolate chosen from the group consisting of Escherichia coli,Cyclospora cayetanensis, Sarcocystis hominis, Eimeria praecox, Entamoebahistolytica, Cryptosporidium parvum and at least one of Cryptosporidiummuris, Cryptosporidium baileyi, Cryptosporidium serpentis andCryptosporidium wrairi. Cryptosporidium parvum and Cryptosporidium murisisolates are available from Waterborne Incorporated (Part No. P102 forCryptosporidium parvum and Part No. P104 for Cryptosporidium muris). Aset of negative control samples is included to determine backgroundlevels.

Each sample is provided to a 12×75 mm polypropylene tube (Gen-ProbeIncorporated; Cat. No. 2440). Additionally, each tube is provided with0.1 pmol of the hybridization assay probe, 5.0 pmol of each helperprobe, and 100 μl 1× Hybridization Reagent. To permit hybridization, thetubes are incubated at 60° C. in a circulating water bath (PrecisionScientific; Model 260; Cat. No. 51221035) for 30 minutes. Followinghybridization, 300 μl Selection Reagent is added to each tube, and thetubes are incubated at 60° C. in the circulating water bath for 10minutes to hydrolyze acridinium ester labels associated withunhybridized probe. Samples are cooled on ice for 1 minute prior tobeing analyzed in a LEADER® 450i luminometer equipped with automaticinjection of the Detection Reagents. A net RLU value greater than 10,000RLU is considered to be a positive result, and a net RLU value less than10,000 RLU is considered to be a negative result. Net RLU values arebased on the average RLU value of each sample set minus the average RLUvalue for the negative control set (i.e., background signal).

Example 4 Sensitivity of Cryptosporidium parvum Hybridization AssayProbes

This example employed controls having target-containing rRNA transcriptsand water samples containing varying amounts of Cryptosporidium parvumoocysts to demonstrate the sensitivity of an acridinium ester-labeledhybridization assay probe for detecting Cryptosporidium parvum nucleicacid in a sample. The hybridization assay probe was synthesized, asdescribed above, to have the base sequence of SEQ ID NO:13 and toinclude a non-nucleotide linker positioned between nucleotides 9 and 10(reading 5′ to 3′). No helper probes were used in this example.

The sensitivity of this Cryptosporidium parvum hybridization assay probewas determined using a panel of duplicate controls and samples (with oneexception), where the controls included the following: (i) a first setcontaining 95 μl lysis reagent and no transcript in each tube (“negativecontrol”); (ii) a second set containing 85 μl lysis reagent and 10 μltranscript at a concentration of 0.01 fmol/μl in each tube (“firstpositive control”); (iii) a third set containing 85 μl lysis reagent and10 μl transcript at a concentration of 0.1 fmol/μl in each tube (“secondpositive control”); (iv) a fourth set containing 85 μl lysis reagent and10 μl transcript at a concentration of 1.0 fmol/μl in each tube (“thirdpositive control”). The lysis reagent was made up of 1.925 ml lysisbuffer (10 mM Tris, pH 8.0, 10 mM CaCl₂ and 1% (w/v) sodium dodecylsulfate (SDS)) and 105 μl proteinase K at a concentration of 20 mg/ml.

Five duplicate samples were tested, with members of each set containingoocyst quantities of 2×10³, 1×10⁴, 1×10⁵, 1×10⁶ and 5×10⁶, respectively,in 1 ml lysis reagent. Unlike the Isolation and Purification methoddetailed above, Cryptosporidium parvum rRNA in this example was isolatedfrom the samples by first centrifuging each sample in a Eppendorf 1.5 mlmicro test tube (Brinkmann Instruments, Inc.; Cat. No. 22 60 002 8) at9,000 rpm for 5 minutes using an Eppendorf microcentrifuge (BrinkmannInstruments, Inc.; Cat. No. 22 62 120-3). All but 100 μl of thesupernatant was removed from each tube, and the samples were againcentrifuged at 9,000 rpm for 2 minutes in the microcentrifuge.Supernatants were removed so as not to disturb the pellets, which wereresuspended in 100 μl lysis reagent.

The samples and controls were all provided with 100 μl Oil Reagent andincubated at 42° C. in a circulating water bath (Lauda Dr. R. WobserGmbH & Co. KG; Model No. M20-S) for 60 minutes. The samples were thentransferred to a dry heat bath (Gen-Probe Incorporated; Cat. No. 4006)and incubated at 95° C. for 90 minutes. Afterwards, each tube received100 μl probe mix prepared by combining 2.2 ml 1× Hybridization Reagentand 22 μl of the hybridization assay probe at a concentration of 100fmol/μl. The contents of the tubes were briefly vortexed and againincubated at 60° C. in the circulating water bath for 30 minutes topermit hybridization of probe to target rRNA.

Following hybridization, 300 μl Selection Reagent was added to eachtube, and the tubes were briefly vortexed before another incubation at60° C. in the circulating water bath for 8 minutes to hydrolyzeacridinium ester labels associated with unhybridized probe. The contentsof the tubes were then cooled at room temperature for 5 minutes prior tobeing analyzed in a LEADER® 450hc luminometer (Gen-Probe Incorporated)equipped with automatic injection of the Detection Reagents. The RLU andpercent coefficient of variation (CV) values from this experiment arepresented in Table 1 below, where “net RLU” is the average RLU of thenegative control, positive control or sample minus the average RLU forthe negative control.

TABLE 1 Hybridization of Probe to Varying Concentrations of TargetNucleic Acid Present in Positive Controls and Isolated from VaryingAmounts of Cryptosporidium parvum Oocysts Average Average Sample RLU RLUNet RLU % CV Negative Control 2,449 2,446 0 0% (0 fmol Target) 2,443First Positive Control 6,147 7,372 4,926 23%  (0.1 fmol Target) 8,597Second Positive Control 43,075 43,075 40,629 n/a (1 fmol Target) ThirdPositive Control 257,757 266,061 263,615 4% (10 fmol Target) 274,364First Sample 4,263 4,356 1,190 3% (2 × 10³ Oocysts) 4,448 Second Sample9,623 9,702 7,256 1% (1 × 10⁴ Oocysts) 9,781 Third Sample 39,267 39,46837,022 1% (1 × 10⁵ Oocysts) 39,668 Fourth Sample 503,780 444,811 442,36519%  (1 × 10⁶ Oocysts) 385,842 Fifth Sample 461,603 457,043 454,597 1%(5 × 10⁶ Oocysts) 452,482

The results of this experiment suggest an rRNA copy number ofapproximately 6,000 per oocyst, although this value might have beenhigher following the novel and more effective Isolation and Purificationmethod described above. This copy number was calculated by linearinterpolation using the algorithm y=mx+b, where “y” is the number ofrelative light units, “m” is the slope, “x” is the number of oocysts and“b” is the y intercept, with 1 fmol of positive control containingapproximately 6.02×10⁸ transcript copies. FIG. 1 is an oocyst titrationgraph plotting “Average Net RLU” versus “Oocysts” determined byhemocytometer counting, which provides an indication of the oocyst loadnecessary detect the presence of Cryptosporidium parvum in a testsample. These results indicate that the presence of Cryptosporidiumparvum organisms in a water sample can be directly detected withouthaving to perform a preliminary amplification step to generatesufficient target sequences.

Example 5 Amplification and Detection of Cryptosporidium parvum NucleicAcid

This example illustrates the use of a Cryptosporidium parvumhybridization assay probe to detect product of a nucleic acidamplification. In particular, a Cryptosporidium parvum hybridizationassay probe having the base sequence of SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:8 or SEQ ID NO:10 is synthesized, as described above, to include anon-nucleotide linker positioned as follows: (i) between nucleotides 18and 19 for the base sequence of SEQ ID NO:5 (reading 5′ to 3′); (ii)between nucleotides 13 and 14 for the base sequence of SEQ ID NO:7(reading 5′ to 3′); (iii) between nucleotides 16 and 17 for the basesequence of SEQ ID NO:8 (reading 5′ to 3′); and (iv) between nucleotides11 and 12 for the base sequence of SEQ ID NO: 10 (reading 5′ to 3′).Except for the first nucleotide (reading 5′to 3′), which is substitutedwith the corresponding deoxynucleotide, each nucleotide of thehybridization assay probe having the base sequence of SEQ ID NO: 10 is aribonucleotide modified to include 2′-O-methyl substitution to theribofuranosyl moiety.

These hybridization assay probes are the same sense as theCryptosporidium target rRNA and are used to detect product of atranscription-mediated amplification. Procedures for performing atranscription-mediated amplification are described supra and by Kacianet al. in U.S. Pat. Nos. 5,399,491 and 5,480,784. In addition, thesehybridization assay probes are the opposite sense of the hybridizationassay probes having the base sequences of SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:16 and SEQ ID NO:18, respectively, of Example 3, which arebelieved to be specific for nucleic acid from Cryptosporidium parvumorganisms. Accordingly, the hybridization assay probes of this exampleare expected to be specific for nucleic acid derived fromCryptosporidium parvum organisms.

Ribosomal RNA from Cryptosporidium parvum is amplified using one of thefollowing promoter-primer/primer combinations when the hybridizationassay probe has the base sequence of SEQ ID NO:5, SEQ ID NO:7 or SEQ IDNO:10 (modified as indicated): (i) a promoter-primer having a 5′ endpromoter base sequence of SEQ ID NO:69 and a 3′ end sensetemplate-specific base sequence of SEQ ID NO:59, and a primer having anantisense template-specific base sequence of SEQ ID NO:45; (ii) apromoter-primer having a 5′ end promoter base sequence of SEQ ID NO:69and a 3′ end sense template-specific base sequence of SEQ ID NO:59, anda primer having an antisense template-specific base sequence of SEQ IDNO:46; (iii) a promoter-primer having a 5′ end promoter base sequence ofSEQ ID NO:69 and a 3′ end sense template-specific base sequence of SEQID NO:60, and a primer having an antisense template-specific basesequence of SEQ ID NO:45; and (iv) a promoter-primer having a 5′ endpromoter base sequence of SEQ ID NO:69 and a 3′ end sensetemplate-specific base sequence of SEQ ID NO:60, and a primer having anantisense template-specific base sequence of SEQ ID NO:46. And, when thehybridization assay probe has the base sequence of SEQ ID NO:8, rRNAfrom Cryptosporidium parvum is amplified using a promoter-primer havinga 5′ end promoter base sequence of SEQ ID NO:69 and a 3′ end sensetemplate-specific base sequence of SEQ ID NO:62, and a primer having anantisense template-specific base sequence of SEQ ID NO:49. ACryptosporidium parvum isolate is available from Waterborne Incorporatedas Part No. P102.

Amplification is carried out in 12×75 mm polypropylene tubes (Gen-ProbeIncorporated; Cat. No. 2440), each containing 0 amol, 0.2 amol or 2.0amol of Cryptosporidium parvum rRNA in duplicate sets, 15 pmolpromoter-primer, 15 pmol primer, 25 μl Amplification Reagent, anddistilled, deionized water to bring the total volume in each tube to 75μl. Each sample receives 200 μl Oil Reagent and is incubated at 95° C.in a dry heat bath (Gen-Probe Incorporated; Cat. No. 4006) for 10minutes. The samples are then transferred to a circulating water bath(Lauda Dr. R. Wobser GmbH & Co. KG; Model No. M20-S) and incubated at42° C. for 5 minutes before adding 25 μl of reconstituted Enzyme Reagentto each tube. Following a 60 minute incubation at 42° C. in thecirculating water bath, 100 μl 1× Hybridization Reagent containing 100fmol of the hybridization assay probe is added to each sample, thesamples are incubated for 30 minutes at 60° C. in the circulating waterbath, and signal from annealed hybridization assay probe is detected inthe manner described in Example 3. Sample sets with an average RLU valuegreater than 10-fold the average RLU value for the negative control (0amol target rRNA) indicate amplification of the target rRNA, and samplesets with an average RLU value less than 10-fold the average RLU for thenegative control indicate no amplification of the target rRNA.

Example 6 Amplification of a Cryptosporidium parvum Target Transcript

This example illustrates amplification and detection of aCryptosporidium parvum rRNA transcript sequence at variousconcentrations. In particular, a Cryptosporidium parvum hybridizationassay probe having the base sequence of SEQ ID NO:5 was synthesized, asdescribed above, to include a non-nucleotide linker positioned betweennucleotides 18 and 19 (reading 5′ to 3′). This hybridization assay probewas the same sense as the Cryptosporidium target rRNA and was used todetect product of a transcription-mediated amplification. Procedures forperforming a transcription-mediated amplification are described supraand by Kacian et al. in U.S. Pat. Nos. 5,399,491 and 5,480,784. Inaddition, this hybridization assay probe was the opposite sense of thehybridization assay probe having the base sequence of SEQ ID NO:13 ofExample 3, which is believed to be specific for nucleic acid fromCryptosporidium parvum organisms. Accordingly, the hybridization assayprobe of this example was expected to be specific for nucleic acidderived from Cryptosporidium parvum organisms.

For this example, two different promoter-primer/primer combinations wereemployed. The first promoter-primer/primer combination included apromoter-primer having a 5′ end promoter base sequence of SEQ ID NO:69and a 3′ end sense template-specific base sequence of SEQ ID NO:59, anda primer having an antisense template-specific base sequence of SEQ IDNO:46 (“primer set one”). The second of these promoter-primer/primercombinations included a promoter-primer having a 5′ end promoter basesequence of SEQ ID NO:69 and a 3′ end sense template-specific basesequence of SEQ ID NO:59, and a primer having an antisensetemplate-specific base sequence of SEQ ID NO:45 (“primer set two”). Theworking stock for each primer set was 200 μl distilled, deionized watercontaining primer and promoter-primer, each at a concentration of 15pmol/μl.

The working stocks for each primer set were then used to prepare twoamplification solutions. The first of these solutions was prepared bycombining 375 μl Amplification Reagent, 15 μl primer set one workingstock and 585 μl distilled, deionized water (“first amplificationsolution”). The second of these solutions was prepared by combining 375μl Amplification Reagent, 15 μl primer set two working stock and 585 μldistilled, deionized water (“second amplification solution”).

The probe stock was made up of the hybridization assay probe at aconcentration of 100 fmol/μl in approximately 200 μl 1× HybridizationReagent. From this stock, the probe mix was prepared by combining 30 μlprobe stock and 3 ml 2× Hybridization Reagent. This volume of probestock was sufficient for up to 30 samples.

Transcript dilutions were made to prepare transcript stocks havingconcentrations of 10 amol/μl, 1 amol/μl, 0.1 amol/μl, 0.01 amol/μl and0.001 amol/μl in distilled, deionized water. (Each amol of transcriptstock contained approximately 600,000 copies of the transcript.) Fromeach of these stock concentrations, a set of four Eppendorf 1.5 ml microtest tubes (Brinkmann Instruments, Inc.; Cat. No. 22 60 002 8) wasprepared, each containing 10 μl transcript stock solution. The contentsof each of two tubes from each tube set were combined with 65 μl of thefirst amplification solution, while the contents of each of the othertwo tubes of each tube set were combined with 65 μl of the secondamplification solution. Also included were four negative control tubes(0 amol target), each containing 10 μl of distilled, deionized water.Two of these negative controls received 65 μl of the first amplificationsolution, while the other two negative controls received 65 μl of thesecond amplification solution.

Each tube received 200 μl Oil Reagent (both samples and controls), andthe tubes were incubated at 95° C. in a dry heat bath (Gen-ProbeIncorporated; Cat. No. 4006) for 5 minutes. The tubes were thentransferred to a circulating water bath (Lauda Dr. R. Wobser GmbH & Co.KG; Model No. M20-S) for a 5 minute incubation at 42° C. before adding25 μl of reconstituted Enzyme Reagent to each tube. The tubes were thenmixed gently and incubated for an additional 60 minutes at 42° C. in thecirculating water bath. Following this third incubation, 100 μl probemix was added to each tube, the tubes were vortexed, and subjected to afourth incubation at 60° C. for 30 minutes in the circulating waterbath. Each tube was then provided 300 μl Selection Reagent, vortexed,incubated at 60° C. for 10 minutes in the circulating water bath, andcooled on ice water for 1 minute. Signal from annealed hybridizationassay probe was detected in the manner described in Example 3, exceptthat a LEADER® 450hc luminometer was used instead of a LEADER® 450iluminometer, and the results are presented in Table 2 below. Sample setswith an average RLU value greater than 10-fold the average RLU value forthe negative control (0 amol target rRNA) indicated amplification of thetarget rRNA, and sample sets with an average RLU value less than 10-foldthe average RLU for the negative control indicated no amplification ofthe target rRNA.

TABLE 2 Hybridization of Probe to Amplification Product Generated fromVarying Initial Concentrations of Cryptosporidium parvum Target NucleicAcid Using Different Primer Sets % Sample RLU Average Average CV PrimerNegative Control 5,507 5,524 0  0% Set (0 amol Target) 5,540 One FirstSample 4,399,434 4,319,893 4,314,370  3% (100 amol Target) 4,240,352Second Sample 3,391,820 3,411,497 3,405,974  1% (10 amol Target)3,431,174 Third Sample 2,061,629 1,972,710 1,967,186  6% (1 amol Target)1,883,790 Fourth Sample 328,577 307,112 301,588 10% (0.1 amol Target)285,646 Fifth Sample 48,749 44,485 38,961 14% (0.01 amol Target) 40,220Primer Negative Control 6,169 8,380 0 37% Set (0 amol Target) 10,590 TwoFirst Sample 11,913 23,292 14,912 69% (100 amol Target) 34,670 SecondSample 10,533 6,810 −1,570 77% (10 amol Target) 3,087 Third Sample10,394 10,346 1,967  1% (1 amol Target) 10,298 Fourth Sample 8,907 8,849470  1% (0.1 amol Target) 8,791 Fifth Sample 9,295 8,174 −206 19% (0.01amol Target) 7,053

The results of this experiment demonstrate that primer set one is usefulunder these conditions for amplifying the target sequence and can beused to detect the presence of target rRNA from Cryptosporidium parvumpresent in a sample having a copy number at least as low as 6,000 (whichis believed to represent a fraction of the rRNA present in an oocyst).FIG. 2 is an amplification graph plotting “Average Net RLU” versus“Target,” which provides an indication of the initial rRNA copy numberneeded to detect the presence of Cryptosporidium parvum organisms in atest sample following a transcription-mediated amplification. From thisexample, it is would appear that 1,000 copies of rRNA (less than theamount expected to be present in a single Cryptosporidium parvum oocyst)is sufficient to detect the presence of Cryptosporidium parvum organismsin a test sample when sample rRNA is amplified by performing atranscription-mediated amplification. Through routine experimentation,it is believed that the conditions and concentrations set forth in thisexample could be optimized so that primer set two could likewise amplify(as defined) the target sequence.

Example 7 Amplification of a Cryptosporidium parvum Target Transcript inthe Presence of rRNA from a Non-Target Organism

This example illustrates amplification and detection of aCryptosporidium parvum rRNA transcript sequence in the presence ofCryptosporidium muris rRNA at various concentrations of each. Inparticular, a Cryptosporidium parvum hybridization assay probe havingthe base sequence of SEQ ID NO:5 was synthesized, as described above, toinclude a non-nucleotide linker positioned between nucleotides 18 and 19(reading 5′ to 3′). This hybridization assay probe was the same sense asthe Cryptosporidium target rRNA and was used to detect product of atranscription-mediated amplification. Procedures for performing atranscription-mediated amplification are described supra and by Kacianet al. in U.S. Pat. Nos. 5,399,491 and 5,480,784. In addition, thishybridization assay probe was the opposite sense of the hybridizationassay probe having the base sequence of SEQ ID NO:13 of Example 3, whichis believed to be specific for nucleic acid from Cryptosporidium parvumorganisms. Accordingly, it would be expected that the hybridizationassay probe of this example would be specific for nucleic acid derivedfrom Cryptosporidium parvum organisms.

In this example, a total of 20 12×75 mm polypropylene tubes (Gen-ProbeIncorporated; Cat. No. 2440) were set up in a matrix format to containthe amounts of Cryptosporidium parvum transcript rRNA andCryptosporidium muris rRNA indicated in Table 3 below. TheCryptosporidium parvum oocysts used to generate the transcript of thisexample were obtained from Waterborne Incorporated under Part No. P102.The Cryptosporidium muris oocysts were obtained from a private source,however, a Cryptosporidium muris isolate is available from WaterborneIncorporated under Part No. 104. For Cryptosporidium muris, duplicatesets of tubes were provided with 0 amol, 0.2 amol, 2 amol, 20 amol and200 amol of Cryptosporidium muris sample rRNA. Each member of these setsof tubes was then matched with one of two Cryptosporidium parvum tubeshaving either 0 amol or 2 amol Cryptosporidium parvum transcript rRNA.The promoter-primer/primer combination provided to each tube included apromoter-primer having a 5′ end promoter base sequence of SEQ ID NO:69and a 3′ end sense template-specific base sequence of SEQ ID NO:59, anda primer having an antisense template-specific base sequence of SEQ IDNO:46. This primer set was used to prepare an amplification solutionmade up of 750 μl Amplification Reagent and 30 μl of a primer workingstock containing 15 pmol/μl each of the promoter-primer and the primerin distilled, deionized water.

Each tube was provided 25 μl of the amplification solution, and thenon-control tubes were provided with 10 μl of the appropriate dilutionof sample rRNA. The total volume of all tubes was brought up to 75 μlwith distilled, deionized water. The tubes were separately provided 200μl Oil Reagent and then incubated at 95° C. in a dry heat bath(Gen-Probe Incorporated; Cat. No. 4006) for 10 minutes, followed by a 5minute incubation at 42° C. in a circulating water bath (Lauda Dr. R.Wobser GmbH & Co. KG; Model No. M20-S). After adding 25 μl ofreconstituted Enzyme Reagent to each tube, the tubes were mixed gentlyand incubated for an additional 60 minutes at 42° C. in the circulatingwater bath. Following this third incubation, 100 μl probe mix was addedto each tube, the probe mix being comprised of 1 pmol/ml of thehybridization assay probe in 1× Hybridization Reagent. The tubes werethen vortexed and subjected to a fourth incubation at 60° C. for 30minutes in the circulating water bath. Each tube was then provided 300μl Selection Reagent, vortexed, incubated at 60° C. for 10 minutes inthe circulating water bath, and then cooled on ice for 1 minute. Signalfrom annealed hybridization assay probe was detected in the mannerdescribed in Example 3, except that a LEADER® 450hc luminometer was usedinstead of a LEADER® 450i luminometer, and the results are presented inTable 3 below. Samples with an RLU value greater than 10-fold the RLUvalue for the negative control (0 amol target rRNA and 0 amol non-targetrRNA) indicated amplification of the target rRNA or non-target rRNA, andsamples with an RLU value less than 10-fold the RLU for the negativecontrol indicated no amplification of the target rRNA or non-targetrRNA.

TABLE 3 Hybridization of Probe to Amplification Product Generated fromVarying Initial Concentrations of Cryptosporidium parvum TargetTranscript in the Presence of Varying Concentrations of Cryptosporidiummuris rRNA C. muris C. parvum Fold (amol rRNA) (amol rRNA) RLUBackground 0 0 24,215 1 0 2 3,908,824 161 0.2 0 25,449 1 0.2 2 3,272,793135 2 0 38,866 2 2 2 3,786,620 156 20 0 81,328 3 20 2 2,408,251 99 200 0116,466 5 200 2 4,359,656 180

The results of this experiment demonstrate that the primer set is usefulfor amplifying a target sequence present in nucleic acid fromCryptosporidium parvum in the presence of potentially interferingnucleic acid from a closely-related, non-targeted organism.Additionally, the data demonstrates that the hybridization assay probespecifically hybridizes to amplicon derived from Cryptosporidium parvumnucleic acid.

1. A method for obtaining purified RNA from viable oocysts, the methodcomprising the steps of: a) centrifuging a fluid sample containingoocysts at a speed and for a period of time sufficient to concentratethe oocysts within a vessel containing the fluid sample; b) removing asupernatant formed in the vessel during the centrifuging step; c)resuspending the concentrated oocysts in a buffered solution; d)agitating the buffered solution in the presence of zirconia/silicaparticles at a rate and for a period of time sufficient to lyse theoocysts and release RNA therefrom; e) immobilizing the released RNA onan RNA-binding filter; f) purifying the released RNA by washing thefilter one or more times with a buffered solution to remove oocystcomponents other than the released RNA; and g) removing the purified RNAfrom the filter.
 2. The method of claim 1, wherein said oocysts includeCryptosporidium organisms.
 3. The method of claim 2, wherein saidCryptosporidium organisms include Cryptosporidium parvum.
 4. The methodof claim 1, wherein said centrifuging step is performed at about 7000revolutions per minute for approximately 5 minutes.
 5. The method ofclaim 1, wherein the buffered solution includes a chaotropic agentcapable of inactivating endogenous ribonucleases released from theoocysts.
 6. The method of claim 5, wherein the chaotropic agent isguanidinium thiocyanate.
 7. The method of claim 1, wherein the agitatingstep is performed by oscillating or vortexing the buffered solution. 8.The method of claim 1, wherein the particles have a density of about 3.7g/cc.
 9. The method of claim 1, wherein the particles have a generallyspherical shape.
 10. The method of claim 9, wherein the particles havean average diameter in the range of about 0.1 to about 2.5 mm.
 11. Themethod of claim 9, wherein the particles have an average diameter in therange of about 0.5 to about 1 mm.
 12. The method of claim 9, wherein theparticles have an average diameter of about 1 mm.
 13. The method ofclaim 1, wherein the filter is a silica-based matrix.
 14. The method ofclaim 1, wherein the oocyst components include proteins and DNA.